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Accelerated endometrial maturation in the luteal phase of cycles utilizing controlled ovarian hyperstimulation: impact of gonadotropin-releasing hormone agonists versus antagonists
FERTILITY AND STERILITY威 VOL. 82, NO. 1, JULY 2004 Copyright ©2004 American Society for Reproductive Medicine Published by Elsevier Inc. Printed on acid-free paper in U.S.A.
Accelerated endometrial maturation in the luteal phase of cycles utilizing controlled ovarian hyperstimulation: impact of gonadotropin-releasing hormone agonists versus antagonists Peyman Saadat, M.D., Robert Boostanfar, M.D., Cristin C. Slater, M.D., David E. Tourgeman, M.D., Frank Z. Stanczyk, Ph.D., and Richard J. Paulson, M.D. Keck School of Medicine of the University of Southern California, Los Angeles, and Good Samaritan Hospital, Los Angeles, California
Objective: To evaluate the endometrium obtained during the luteal phase of controlled ovarian hyperstimulation (COH) cycles utilizing gonadotropin-releasing hormone (GnRH) antagonists, and to compare these findings with those obtained in cycles utilizing a GnRH agonist and with artificial cycles among recipients. Design: Prospective evaluation of oocyte donors. Setting: University-based in vitro fertilization (IVF) center. Patient(s): Fifteen oocyte donors undergoing standard COH were enrolled in 1 of 3 COH groups, and 40 recipients of oocyte donation were used as a control group. Intervention(s): Controlled ovarian hyperstimulation and endometrial biopsy. Main Outcome Measure(s): Histological dating of endometrial biopsies, serum estradiol (E2) and progesterone levels. Result(s): On the day of oocyte retrieval, endometrial maturation was advanced by an average of 5.8 ⫾ 0.4 days in the antagonist group and 5.9 ⫾ 0.7 days in the agonist group. This advancement persisted on day 7 postoocyte retrieval. Serum progesterone levels were elevated before human chorionic gonadotropin (hCG) administration, but remained similar in both groups. Conclusion(s): Controlled ovarian hyperstimulation is associated with elevated progesterone levels in the late follicular phase and accelerated endometrial maturation in the subsequent luteal phase. No significant differences exist between preretrieval serial serum progesterone levels and luteal phase endometrial histology between cycles utilizing GnRH agonists or antagonists. (Fertil Steril威 2004;82:167–71. ©2004 by American Society for Reproductive Medicine.) Key Words: Controlled ovarian hyperstimulation, GnRH antagonist, GnRH agonist, endometrial maturation, progesterone, oocyte donation Received April 7, 2003; revised and accepted November 25, 2003. Reprint requests: Peyman Saadat, M.D., Department of Obstetrics and Gynecology, Women’s and Children’s Hospital, 1240 North Mission Road, Room 8K9, Los Angeles, California 90033 (FAX: 323226-2850; E-mail: [email protected]). 0015-0282/04/$30.00 doi:10.1016/j.fertnstert.2003. 11.050
The introduction of gonadotropin-releasing hormone (GnRH) antagonists as an alternative to GnRH agonists for ovulation inhibition during controlled ovarian hyperstimulation (COH) has added to the pharmacological armamentarium of the practice of assisted reproductive technology (ART). Early reports revealed that GnRH antagonists were effective in preventing the LH rise during ovarian stimulation for IVF (1– 4). Their clinical efficacy was subsequently confirmed by several large studies (5–9). When compared with protocols using GnRH agonists,
COH cycles utilizing GnRH antagonists have been shown to successfully suppress LH, require lower total amounts of follicle-stimulating hormone (FSH), and result in lower rates of ovarian hyperstimulation syndrome (OHSS). In addition, despite significantly lower serum estradiol (E2) levels on the day of human chorionic gonadotropin (hCG) administration and the number of oocytes retrieved, fertilization rates and embryo quality appear to be similar between COH cycles utilizing GnRH agonists when compared with cycles using 167
GnRH antagonists. However, a trend toward decreasing pregnancy rates and ongoing pregnancies has been observed (5–9). Because embryo quality appears unaffected, and yet per-embryo implantation rates are diminished, it has been suggested that the effects of GnRH antagonists on the latefollicular phase estrogen and progesterone levels may have an impact on endometrial maturation (10) and consequently implantation. It has been previously reported by our group as well as others that advanced endometrial maturation occurs during the luteal phase of cycles utilizing COH. Elevation of serum progesterone during the late-follicular phase of these cycles appears to be the inciting cause of this precocious endometrial development. Premature luteinization has been demonstrated by histology and endometrial ultrastructure in COH cycles as compared with natural cycles and artificial cycles of recipients of oocyte donation (11, 12); however, the impact of COH utilizing GnRH antagonists on the endometrium has not yet been characterized. The objective of our study was to prospectively measure ovarian steroid production in the late follicular phase and its effects on endometrial histomorphology during the subsequent luteal phase among subjects undergoing COH utilizing GnRH agonists or antagonists.
MATERIALS AND METHODS We prospectively studied 15 oocyte donors, age 20 to 32 years old, who were participants in our oocyte donation program, between July and November of 2001. All donors underwent standard COH. Subjects underwent standard screening for oocyte donors and all signed informed consents before enrollment in the study. This study was approved by the Institutional Review Board of the Good Samaritan Hospital Los Angeles. Participants were prospectively assigned to one of two COH protocols. The first group (n ⫽ 10) received one of two GnRH antagonists (five patients on cetrorelix or ganirelix each) as described previously (9). Briefly, subjects were given oral contraceptives for 2 to 3 weeks. Stimulation with hMG was begun on day 4 after the last oral contraceptive pill regardless of bleeding history. Daily GnRH antagonist was added when the lead follicle reached a maximum diameter of 14 mm. Human chorionic gonadotropin was administered when two lead follicles reached a mean diameter of 18 mm. The second group (n ⫽ 5) was assigned to the long GnRH agonist protocol (leuprolide acetate) as described previously (13). Serial serum E2 and progesterone levels were measured throughout ovarian stimulation. Estradiol was measured using extraction and radioimmunoassay. Progesterone was determined using a chemilumenscent assay. An endometrial biopsy was obtained at the time of oocyte retrieval (36 hours after hCG) and again 7 days later. 168
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FIGURE 1 Assessment of endometrial maturation by histomorphology (mean ⫾ SD postovulatory day) in the agonist and antagonist groups. (P⬎.05 agonists vs. antagonists). (red bar) Antagonist group (n ⫽ 10), (blue bar) Agonist group (n ⫽ 5).
Saadat. Accelerated endometrial maturation in GnRH antagonist cycles. Fertil Steril 2004.
A group of 40 recipients of oocyte donation undergoing a mock cycle using the standard estradiol/progesterone protocol (14) were used as a concurrent endometrial histologic control. Endometrial biopsy was performed on the morning of day 7 of progesterone. An experienced endometrial histopathologist blinded to the treatment groups read all endometrial biopsies. The endometrial dating was assessed by the criteria of Noyes et al. (15). We considered a difference of 2 or more days in the histological dating as representing a significant difference between agonist and antagonist cycles. We anticipated a standard deviation of ⫾ 1 day in the histological dating. Therefore, to detect a difference of 2 days, power analysis
FIGURE 2 Assessment of endometrial maturation by histomorphology (mean ⫾ SD postovulatory day) among patients undergoing COH (agonist or antagonist) and among recipients of oocyte donation undergoing mock cycles using standard estradiol/ progesterone protocol. (P⬍.05 COH vs. recipients). (red bar) Antagonist group (n ⫽ 10), (blue bar) Agonist group (n ⫽ 5), (green bar) Recipient (n ⫽ 40).
Saadat. Accelerated endometrial maturation in GnRH antagonist cycles. Fertil Steril 2004.
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Vol. 82, No. 1, July 2004
TABLE 1 Mean serum estradiol (pg/mL) values.a hCG-4
hCG-3
hCG-2
Day of hCG
Oocyte retrieval
439 ⫾ 231 230 ⫾ 84
722 ⫾ 326 654 ⫾ 294
973 ⫾ 328 1,203 ⫾ 372
1736 ⫾ 894 2,671 ⫾ 1,169
788 ⫾ 248 1,172 ⫾ 545
Groupb Antagonist (n ⫽ 10) Agonist (n ⫽ 5)
Note: hCG ⫽ human chorionic gonadotropin. Results reported as mean value ⫾ SD. b Differences between the agonist and antagonist groups not statistically significant. a
Saadat. Accelerated endometrial maturation in GnRH antagonist cycles. Fertil Steril 2004.
indicated that we needed five patients in each group. Data were analyzed by using analysis of variance and unpaired Student’s t-test.
RESULTS At the time of oocyte retrieval, which corresponds to day 0 of the luteal phase, the average histomorphological dating of the endometrium was assessed as postovulatory day 5.9 ⫾ 0.7 days in the agonist group and 5.8 ⫾ 0.4 days in the antagonist group (Fig. 1). This advancement in the endometrial maturation persisted similarly in both groups on day 7 postoocyte retrieval. The average endometrial dating was reported as 9.6 ⫾ 0.9 days postovulation in the antagonist group and 10.2 ⫾ 0.8 days postovulation in the agonist group on the biopsy performed 7 days after oocyte retrieval. This advancement demonstrated statistically significantly greater progression than the endometrial maturation of the recipient group (postovulatory day 5.2 ⫾ 0.3) obtained on the morning of day 7 of progesterone during the mock stimulation cycle (Fig. 2). Serum E2 and progesterone levels are depicted in Table 1 and Table 2. Serum E2 levels initially rose faster in the COH cycles utilizing a GnRH antagonist compared with cycles using an agonist; however, this trend reversed 2 days before hCG administration (Table 1). No significant differences between E2 levels in the two groups were noted. Analo-
gously, no significant differences were noted in serum progesterone levels between the two groups throughout the cycle (Table 2). A gradual increase in the serum progesterone levels was noted in both groups; a statistically significant increase over baseline was noted 2 days before hCG in the agonist group and on the day of hCG administration in the antagonist group.
DISCUSSION Gonadotropin-releasing hormone antagonists are commonly used in daily IVF practice to inhibit ovulation during COH. Their clinical efficacy and ease of use has proven them to be an effective and useful alternate to GnRH agonists (5–9). However, trends toward lower pregnancy rates have been observed, leading some investigators to postulate various explanations for these trends. A decrease in endometrial receptivity presents one plausible mechanism for this trend. Whereas multiple studies have previously evaluated the endometrium of patients undergoing COH with GnRH agonists (11, 12, 16 –19), none have compared these results with cycles utilizing antagonists. In this preliminary study, we found that COH resulted in advancement in the endometrial maturation regardless of whether the subjects received a GnRH agonist or an antagonist. Advancement in the endometrial maturation has been demonstrated by us as well as other authors not only histo-
TABLE 2 Mean serum progesterone (ng/mL) values.a Groupb Antagonist (n ⫽ 10) Agonist (n ⫽ 5)
hCG-4
hCG-3
hCG-2
Day of hCG
Oocyte retrieval
0.6 ⫾ 0.2 0.6 ⫾ 0.3
0.8 ⫾ 0.3 0.9 ⫾ 0.3
0.8 ⫾ 0.3 1.5 ⫾ 0.8c
1.4 ⫾ 0.5c 1.9 ⫾ 0.8
10.5 ⫾ 4.2 13.3 ⫾ 8.0
Note: hCG ⫽ human chorionic gonadotropin. Results reported as mean value ⫾ SD. b Differences between agonist and antagonist groups not statistically significant. c P⬍.05 vs. baseline. a
Saadat. Accelerated endometrial maturation in GnRH antagonist cycles. Fertil Steril 2004.
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logically (12, 16, 17), but also by the assessment of endometrial ultrastructure as noted by scanning electron microscopy (11, 12, 18, 19). In examining the endometrium of the patients studied, we found a profound advancement in maturation at the time of oocyte retrieval in GnRH agonist and antagonist cycles. The nearly 6-day endometrial advancement observed on day 0 persisted an average of 4 or more days over the 1 week biopsy interval. Thus, 7 days later the histological dating was found to be consistent with postovulatory day 10. This advancement in the endometrial dating was statistically significant compared with recipients. No significant differences occurred in histological dating in agonist cycles compared with antagonist cycles. One of the weaknesses of this type of analysis is the relative lack of precision and accuracy of histological dating. However, the pathologist was blinded to the treatment group in all cases and, therefore, no consistent bias would be expected to have been introduced. Supraphysiological E2 levels were observed in all COH cycles utilizing GnRH analogs (agonist and antagonist). Some controversy surrounds the question of whether or not such excessive elevation in serum E2 may decrease endometrial receptivity. Whereas some investigators have found a decrease in implantation rates (20), others have not found high E2 levels to be associated with an adverse effect (21, 22). We found no significant differences between the serum E2 levels between the cycles utilizing GnRH agonists when compared with those using GnRH antagonists. Elevated progesterone levels were also observed in the late follicular phase before the administration of hCG. Serum progesterone levels were markedly elevated at the time of oocyte retrieval 36 hours after hCG administration. Prior studies have suggested that elevation of serum progesterone beyond a certain threshold may be associated with poor outcome in IVF (23, 24). However, others have not found this assessment to be predictive of IVF outcome (25, 26). Whereas elevated progesterone levels do not appear to adversely affect oocyte quality (27), there is little doubt that the endometrium responds by luteinizing prematurely (11, 12, 18, 19). In the present study, serum progesterone levels started increasing 3 days before the hCG administration and rose to superphysiologic levels 2 to 4 days before oocyte retrieval. This elevation in serum progesterone levels was observed in all subjects undergoing COH regardless of the use of antagonists or agonists. The elevated serum progesterone levels achieved in these cycles may be responsible for the marked advancement in endometrial maturation observed in both study groups. In summary, our findings of accelerated endometrial maturation are consistent with previous reports. However, we found no difference between the endometrial maturation in cycles utilizing GnRH agonists or antagonists. Thus, the observed differences in pregnancy rates between cycles utilizing GnRH agonists or antagonists are unlikely to be due to 170
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a difference in the endometrial maturation or receptivity as assessed by histomorphology. References 1. Frydman R, Cornel C, de Ziegler D, Taieb J, Spitz IM, Bouchard P. Prevention of premature luteinizing hormone and progesterone rise with a gonadotropin-releasing hormone antagonist, Nal-Glu, in controlled ovarian hyperstimulation. Fertil Steril 1991;56:923–7. 2. Frydman R, Cornel C, de Ziegler D, Taieb J, Spitz IM, Bouchard P. Spontaneous luteinizing hormone surges can be reliably prevented by the timely administration of a gonadotropin-releasing antagonist (NalGlu) during the late follicular phase. Hum Reprod 1992;7:930 –3. 3. Cassidenti DL, Sauer MV, Paulson RJ, Ditkoff EC, Rivier J, Yen SSC, et al. Comparison of intermittent and continuous use of a gonadotropinreleasing hormone antagonist (Nal-Glu) in in vitro fertilization cycles: a preliminary report. Am J Obstet Gynecol 1991;165:1806 –10. 4. Ditkoff EC, Cassidenti DL, Paulson RJ, Sauer MV, Paul WL, Rivier J, et al. The gonadotropin-releasing hormone antagonist (Nal-Glu) acutely blocks the luteinizing hormone surge but allows for resumption of folliculogenesis in normal women. Am J Obstet Gynecol 1991;165: 1811–7. 5. The Ganirelix Dose-Finding Study Group. A double-blind, randomized, dose-finding study to assess the efficacy of the gonadotrophin-releasing hormone antagonist ganirelix (Org 37462) to prevent premature luteinizing hormone surges in women undergoing ovarian stimulation with recombinant follicle-stimulating hormone (Puregon威). Hum Reprod 1998;13:3023–31. 6. The North American Ganirelix Study Group. Efficacy and safety of ganirelix acetate versus leuprolide acetate in women undergoing controlled ovarian hyperstimulation. Fertil Steril 2001;75:38 –45. 7. de Jong D, Macklon NS, Eijkemans MJC, Mannaerts BMJL, Coelingh Bennink HJT, Fauser BCJM. Dynamics of the development of multiple follicles during ovarian stimulation for in vitro fertilization using recombinant follicle-stimulating hormone (Puregon) and various doses of the gonadotropin-releasing hormone antagonist ganirelix (Orgalutran/ Antagon). Fertil Steril 2001;75:688 –93. 8. Albano C, Felberbaum RE, Smitz J, Riethmuller-Winzen H, Engel J, Diedrich K, et al. Ovarian stimulation with HMG: results of a prospective randomized phase III European study comparing the luteinizing hormone-releasing hormone (LHRH)-antagonist cetrorelix and the LHRH-agonist buserelin. Hum Reprod 2000;15:526 –31. 9. Akman MA, Erden HF, Tosun SB, Bayazit N, Aksoy E, Bahceci M. Comparison of agonistic flare-up protocol and antagonist multiple dose protocol in ovarian stimulation of poor responders: results of a prospective randomized trial. Hum Reprod 2001;16:868 –70. 10. Hernandez ER. Embryo implantation and GnRH antagonists. Embryo implantation: the Rubicon for GnRH antagonists. Hum Reprod 2000; 15:1211–6. 11. Kolb BA, Najmabadi S, Paulson RJ. Ultrastructural characteristics of the luteal phase endometrium in patients undergoing controlled ovarian hyperstimulation. Fertil Steril 1997;67:625–30. 12. Kolb BA, Paulson RJ. The luteal phase of cycles utilizing controlled ovarian hyperstimulation and the possible impact of this hyperstimulation on embryo implantation. Am J Obstet Gynecol 1997;67:949 –51. 13. Paulson RJ, Hatch IE, Lobo RA, Sauer MV. Cumulative conception and live birth after oocyte donation: implantations regarding endometrial receptivity. Hum Reprod 1997;12:835–9. 14. Sauer MV, Paulson RJ, Lobo RA. Reversing the natural decline in human fertility. An extended clinical trial of oocyte donation to women of advanced reproductive age. JAMA 1992;268:1275–9. 15. Noyes RW, Herting AT, Rock J. Dating the endometrial biopsy. Fertil Steril 1950;1:3–7. 16. Garcia JE, Acosta AA, Hsiu J-G, Jones HS Jr. Advanced endometrial maturation after ovulation induction with human menopausal gonadotropin/human chorionic gonadotropin for in vitro fertilization. Fertil Steril 1984;41:31–5. 17. Forman RG, Eychenne B, Nessmann C, Frydman R, Robel P. Assessing the early luteal phase in in vitro fertilization cycles: relationships between plasma steroids, endometrial receptors, and endometrial histology. Fertil Steril 1989;51:310 –6. 18. Sundstrom P, Nilsson O, Liedholm P. Scanning electron microscopy of human preimplantation endometrium in normal and clomiphene/ human chorionic gonadotropin-stimulated cycles. Fertil Steril 1983; 40:642–7. 19. Nikas G, Psychoyos A. Uterine pinopods in peri-implantation human endometrium. Clinical relevance. Ann N Y Acad Sci 1997;816:129 –42. 20. Valbuena D, Marin J, de Pablo JL, Remohi J, Pellicer A, Simo´ n C. Increasing levels of estradiol are deleterious to embryonic implantation because they directly affect the embryo. Fertil Steril 2001;76:962–8.
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21. Chenette PE, Sauer MV, Paulson RJ. Very high serum estradiol levels are not detrimental to clinical outcome of in vitro fertilization. Fertil Steril 1990;54:858 –63. 22. Gelety TJ, Buyalos RP. The influence of supraphysilogic estradiol levels on human nidation. J Assist Reprod Genet 1995;12:406 –12. 23. Schoolcraft W, Sinton E, Schlenker T, Huynh D, Hamiltion F, Meldrum DR. Lower pregnancy rate with premature luteinization during pituitary suppression with leuprolide acetate. Fertil Steril 1991;55:563–6. 24. Silverberg KM, Burns WN, Olive DL, Reihl RM, Schenken RS. Serum progesterone levels predict success of in vitro fertilization/embryo transfer in patients stimulated with leuprolide acetate and human menopausal gonadotropins. J Clin Endocrinol Metab 1991;73:797–803.
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25. Bustillo M, Stern JJ, Coulam CB. Serum progesterone at the time of human chorionic gonadotrophin does not predict in-vitro fertilization and embryo transfer. Hum Reprod 1995;10:2862–7. 26. Eldestien MC, Seltman HJ, Cox BJ, Robinson SM, Shaw RA, Muasher SJ. Progesterone levels on the day of human chorionic gonadotropin administration in cycles with gonadotropin-releasing hormone agonist suppression are not predictive of pregnancy outcome. Fertil Steril 1990;54:853–7. 27. Legro RS, Ary BA, Paulson RJ, Stanczyk FZ, Sauer MV. Premature luteinization as detected by elevated serum progesterone is associated with a higher pregnancy rate in donor oocyte in-vitro fertilization. Hum Reprod 1993;8:1506 –11.
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Accelerated endometrial maturation in the luteal phase of cycles utilizing controlled ovarian hyperstimulation: impact of gonadotropin-releasing hormone agonists versus antagonists Peyman Saadat, M.D., Robert Boostanfar, M.D., Cristin C. Slater, M.D., David E. Tourgeman, M.D., Frank Z. Stanczyk, Ph.D., and Richard J. Paulson, M.D. Keck School of Medicine of the University of Southern California, Los Angeles, and Good Samaritan Hospital, Los Angeles, California
Objective: To evaluate the endometrium obtained during the luteal phase of controlled ovarian hyperstimulation (COH) cycles utilizing gonadotropin-releasing hormone (GnRH) antagonists, and to compare these findings with those obtained in cycles utilizing a GnRH agonist and with artificial cycles among recipients. Design: Prospective evaluation of oocyte donors. Setting: University-based in vitro fertilization (IVF) center. Patient(s): Fifteen oocyte donors undergoing standard COH were enrolled in 1 of 3 COH groups, and 40 recipients of oocyte donation were used as a control group. Intervention(s): Controlled ovarian hyperstimulation and endometrial biopsy. Main Outcome Measure(s): Histological dating of endometrial biopsies, serum estradiol (E2) and progesterone levels. Result(s): On the day of oocyte retrieval, endometrial maturation was advanced by an average of 5.8 ⫾ 0.4 days in the antagonist group and 5.9 ⫾ 0.7 days in the agonist group. This advancement persisted on day 7 postoocyte retrieval. Serum progesterone levels were elevated before human chorionic gonadotropin (hCG) administration, but remained similar in both groups. Conclusion(s): Controlled ovarian hyperstimulation is associated with elevated progesterone levels in the late follicular phase and accelerated endometrial maturation in the subsequent luteal phase. No significant differences exist between preretrieval serial serum progesterone levels and luteal phase endometrial histology between cycles utilizing GnRH agonists or antagonists. (Fertil Steril威 2004;82:167–71. ©2004 by American Society for Reproductive Medicine.) Key Words: Controlled ovarian hyperstimulation, GnRH antagonist, GnRH agonist, endometrial maturation, progesterone, oocyte donation Received April 7, 2003; revised and accepted November 25, 2003. Reprint requests: Peyman Saadat, M.D., Department of Obstetrics and Gynecology, Women’s and Children’s Hospital, 1240 North Mission Road, Room 8K9, Los Angeles, California 90033 (FAX: 323226-2850; E-mail: [email protected]). 0015-0282/04/$30.00 doi:10.1016/j.fertnstert.2003. 11.050
The introduction of gonadotropin-releasing hormone (GnRH) antagonists as an alternative to GnRH agonists for ovulation inhibition during controlled ovarian hyperstimulation (COH) has added to the pharmacological armamentarium of the practice of assisted reproductive technology (ART). Early reports revealed that GnRH antagonists were effective in preventing the LH rise during ovarian stimulation for IVF (1– 4). Their clinical efficacy was subsequently confirmed by several large studies (5–9). When compared with protocols using GnRH agonists,
COH cycles utilizing GnRH antagonists have been shown to successfully suppress LH, require lower total amounts of follicle-stimulating hormone (FSH), and result in lower rates of ovarian hyperstimulation syndrome (OHSS). In addition, despite significantly lower serum estradiol (E2) levels on the day of human chorionic gonadotropin (hCG) administration and the number of oocytes retrieved, fertilization rates and embryo quality appear to be similar between COH cycles utilizing GnRH agonists when compared with cycles using 167
GnRH antagonists. However, a trend toward decreasing pregnancy rates and ongoing pregnancies has been observed (5–9). Because embryo quality appears unaffected, and yet per-embryo implantation rates are diminished, it has been suggested that the effects of GnRH antagonists on the latefollicular phase estrogen and progesterone levels may have an impact on endometrial maturation (10) and consequently implantation. It has been previously reported by our group as well as others that advanced endometrial maturation occurs during the luteal phase of cycles utilizing COH. Elevation of serum progesterone during the late-follicular phase of these cycles appears to be the inciting cause of this precocious endometrial development. Premature luteinization has been demonstrated by histology and endometrial ultrastructure in COH cycles as compared with natural cycles and artificial cycles of recipients of oocyte donation (11, 12); however, the impact of COH utilizing GnRH antagonists on the endometrium has not yet been characterized. The objective of our study was to prospectively measure ovarian steroid production in the late follicular phase and its effects on endometrial histomorphology during the subsequent luteal phase among subjects undergoing COH utilizing GnRH agonists or antagonists.
MATERIALS AND METHODS We prospectively studied 15 oocyte donors, age 20 to 32 years old, who were participants in our oocyte donation program, between July and November of 2001. All donors underwent standard COH. Subjects underwent standard screening for oocyte donors and all signed informed consents before enrollment in the study. This study was approved by the Institutional Review Board of the Good Samaritan Hospital Los Angeles. Participants were prospectively assigned to one of two COH protocols. The first group (n ⫽ 10) received one of two GnRH antagonists (five patients on cetrorelix or ganirelix each) as described previously (9). Briefly, subjects were given oral contraceptives for 2 to 3 weeks. Stimulation with hMG was begun on day 4 after the last oral contraceptive pill regardless of bleeding history. Daily GnRH antagonist was added when the lead follicle reached a maximum diameter of 14 mm. Human chorionic gonadotropin was administered when two lead follicles reached a mean diameter of 18 mm. The second group (n ⫽ 5) was assigned to the long GnRH agonist protocol (leuprolide acetate) as described previously (13). Serial serum E2 and progesterone levels were measured throughout ovarian stimulation. Estradiol was measured using extraction and radioimmunoassay. Progesterone was determined using a chemilumenscent assay. An endometrial biopsy was obtained at the time of oocyte retrieval (36 hours after hCG) and again 7 days later. 168
Saadat et al.
FIGURE 1 Assessment of endometrial maturation by histomorphology (mean ⫾ SD postovulatory day) in the agonist and antagonist groups. (P⬎.05 agonists vs. antagonists). (red bar) Antagonist group (n ⫽ 10), (blue bar) Agonist group (n ⫽ 5).
Saadat. Accelerated endometrial maturation in GnRH antagonist cycles. Fertil Steril 2004.
A group of 40 recipients of oocyte donation undergoing a mock cycle using the standard estradiol/progesterone protocol (14) were used as a concurrent endometrial histologic control. Endometrial biopsy was performed on the morning of day 7 of progesterone. An experienced endometrial histopathologist blinded to the treatment groups read all endometrial biopsies. The endometrial dating was assessed by the criteria of Noyes et al. (15). We considered a difference of 2 or more days in the histological dating as representing a significant difference between agonist and antagonist cycles. We anticipated a standard deviation of ⫾ 1 day in the histological dating. Therefore, to detect a difference of 2 days, power analysis
FIGURE 2 Assessment of endometrial maturation by histomorphology (mean ⫾ SD postovulatory day) among patients undergoing COH (agonist or antagonist) and among recipients of oocyte donation undergoing mock cycles using standard estradiol/ progesterone protocol. (P⬍.05 COH vs. recipients). (red bar) Antagonist group (n ⫽ 10), (blue bar) Agonist group (n ⫽ 5), (green bar) Recipient (n ⫽ 40).
Saadat. Accelerated endometrial maturation in GnRH antagonist cycles. Fertil Steril 2004.
Accelerated endometrial maturation in GnRH antagonist cycles
Vol. 82, No. 1, July 2004
TABLE 1 Mean serum estradiol (pg/mL) values.a hCG-4
hCG-3
hCG-2
Day of hCG
Oocyte retrieval
439 ⫾ 231 230 ⫾ 84
722 ⫾ 326 654 ⫾ 294
973 ⫾ 328 1,203 ⫾ 372
1736 ⫾ 894 2,671 ⫾ 1,169
788 ⫾ 248 1,172 ⫾ 545
Groupb Antagonist (n ⫽ 10) Agonist (n ⫽ 5)
Note: hCG ⫽ human chorionic gonadotropin. Results reported as mean value ⫾ SD. b Differences between the agonist and antagonist groups not statistically significant. a
Saadat. Accelerated endometrial maturation in GnRH antagonist cycles. Fertil Steril 2004.
indicated that we needed five patients in each group. Data were analyzed by using analysis of variance and unpaired Student’s t-test.
RESULTS At the time of oocyte retrieval, which corresponds to day 0 of the luteal phase, the average histomorphological dating of the endometrium was assessed as postovulatory day 5.9 ⫾ 0.7 days in the agonist group and 5.8 ⫾ 0.4 days in the antagonist group (Fig. 1). This advancement in the endometrial maturation persisted similarly in both groups on day 7 postoocyte retrieval. The average endometrial dating was reported as 9.6 ⫾ 0.9 days postovulation in the antagonist group and 10.2 ⫾ 0.8 days postovulation in the agonist group on the biopsy performed 7 days after oocyte retrieval. This advancement demonstrated statistically significantly greater progression than the endometrial maturation of the recipient group (postovulatory day 5.2 ⫾ 0.3) obtained on the morning of day 7 of progesterone during the mock stimulation cycle (Fig. 2). Serum E2 and progesterone levels are depicted in Table 1 and Table 2. Serum E2 levels initially rose faster in the COH cycles utilizing a GnRH antagonist compared with cycles using an agonist; however, this trend reversed 2 days before hCG administration (Table 1). No significant differences between E2 levels in the two groups were noted. Analo-
gously, no significant differences were noted in serum progesterone levels between the two groups throughout the cycle (Table 2). A gradual increase in the serum progesterone levels was noted in both groups; a statistically significant increase over baseline was noted 2 days before hCG in the agonist group and on the day of hCG administration in the antagonist group.
DISCUSSION Gonadotropin-releasing hormone antagonists are commonly used in daily IVF practice to inhibit ovulation during COH. Their clinical efficacy and ease of use has proven them to be an effective and useful alternate to GnRH agonists (5–9). However, trends toward lower pregnancy rates have been observed, leading some investigators to postulate various explanations for these trends. A decrease in endometrial receptivity presents one plausible mechanism for this trend. Whereas multiple studies have previously evaluated the endometrium of patients undergoing COH with GnRH agonists (11, 12, 16 –19), none have compared these results with cycles utilizing antagonists. In this preliminary study, we found that COH resulted in advancement in the endometrial maturation regardless of whether the subjects received a GnRH agonist or an antagonist. Advancement in the endometrial maturation has been demonstrated by us as well as other authors not only histo-
TABLE 2 Mean serum progesterone (ng/mL) values.a Groupb Antagonist (n ⫽ 10) Agonist (n ⫽ 5)
hCG-4
hCG-3
hCG-2
Day of hCG
Oocyte retrieval
0.6 ⫾ 0.2 0.6 ⫾ 0.3
0.8 ⫾ 0.3 0.9 ⫾ 0.3
0.8 ⫾ 0.3 1.5 ⫾ 0.8c
1.4 ⫾ 0.5c 1.9 ⫾ 0.8
10.5 ⫾ 4.2 13.3 ⫾ 8.0
Note: hCG ⫽ human chorionic gonadotropin. Results reported as mean value ⫾ SD. b Differences between agonist and antagonist groups not statistically significant. c P⬍.05 vs. baseline. a
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logically (12, 16, 17), but also by the assessment of endometrial ultrastructure as noted by scanning electron microscopy (11, 12, 18, 19). In examining the endometrium of the patients studied, we found a profound advancement in maturation at the time of oocyte retrieval in GnRH agonist and antagonist cycles. The nearly 6-day endometrial advancement observed on day 0 persisted an average of 4 or more days over the 1 week biopsy interval. Thus, 7 days later the histological dating was found to be consistent with postovulatory day 10. This advancement in the endometrial dating was statistically significant compared with recipients. No significant differences occurred in histological dating in agonist cycles compared with antagonist cycles. One of the weaknesses of this type of analysis is the relative lack of precision and accuracy of histological dating. However, the pathologist was blinded to the treatment group in all cases and, therefore, no consistent bias would be expected to have been introduced. Supraphysiological E2 levels were observed in all COH cycles utilizing GnRH analogs (agonist and antagonist). Some controversy surrounds the question of whether or not such excessive elevation in serum E2 may decrease endometrial receptivity. Whereas some investigators have found a decrease in implantation rates (20), others have not found high E2 levels to be associated with an adverse effect (21, 22). We found no significant differences between the serum E2 levels between the cycles utilizing GnRH agonists when compared with those using GnRH antagonists. Elevated progesterone levels were also observed in the late follicular phase before the administration of hCG. Serum progesterone levels were markedly elevated at the time of oocyte retrieval 36 hours after hCG administration. Prior studies have suggested that elevation of serum progesterone beyond a certain threshold may be associated with poor outcome in IVF (23, 24). However, others have not found this assessment to be predictive of IVF outcome (25, 26). Whereas elevated progesterone levels do not appear to adversely affect oocyte quality (27), there is little doubt that the endometrium responds by luteinizing prematurely (11, 12, 18, 19). In the present study, serum progesterone levels started increasing 3 days before the hCG administration and rose to superphysiologic levels 2 to 4 days before oocyte retrieval. This elevation in serum progesterone levels was observed in all subjects undergoing COH regardless of the use of antagonists or agonists. The elevated serum progesterone levels achieved in these cycles may be responsible for the marked advancement in endometrial maturation observed in both study groups. In summary, our findings of accelerated endometrial maturation are consistent with previous reports. However, we found no difference between the endometrial maturation in cycles utilizing GnRH agonists or antagonists. Thus, the observed differences in pregnancy rates between cycles utilizing GnRH agonists or antagonists are unlikely to be due to 170
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a difference in the endometrial maturation or receptivity as assessed by histomorphology. References 1. Frydman R, Cornel C, de Ziegler D, Taieb J, Spitz IM, Bouchard P. Prevention of premature luteinizing hormone and progesterone rise with a gonadotropin-releasing hormone antagonist, Nal-Glu, in controlled ovarian hyperstimulation. Fertil Steril 1991;56:923–7. 2. Frydman R, Cornel C, de Ziegler D, Taieb J, Spitz IM, Bouchard P. Spontaneous luteinizing hormone surges can be reliably prevented by the timely administration of a gonadotropin-releasing antagonist (NalGlu) during the late follicular phase. Hum Reprod 1992;7:930 –3. 3. Cassidenti DL, Sauer MV, Paulson RJ, Ditkoff EC, Rivier J, Yen SSC, et al. Comparison of intermittent and continuous use of a gonadotropinreleasing hormone antagonist (Nal-Glu) in in vitro fertilization cycles: a preliminary report. Am J Obstet Gynecol 1991;165:1806 –10. 4. Ditkoff EC, Cassidenti DL, Paulson RJ, Sauer MV, Paul WL, Rivier J, et al. The gonadotropin-releasing hormone antagonist (Nal-Glu) acutely blocks the luteinizing hormone surge but allows for resumption of folliculogenesis in normal women. Am J Obstet Gynecol 1991;165: 1811–7. 5. The Ganirelix Dose-Finding Study Group. A double-blind, randomized, dose-finding study to assess the efficacy of the gonadotrophin-releasing hormone antagonist ganirelix (Org 37462) to prevent premature luteinizing hormone surges in women undergoing ovarian stimulation with recombinant follicle-stimulating hormone (Puregon威). Hum Reprod 1998;13:3023–31. 6. The North American Ganirelix Study Group. Efficacy and safety of ganirelix acetate versus leuprolide acetate in women undergoing controlled ovarian hyperstimulation. Fertil Steril 2001;75:38 –45. 7. de Jong D, Macklon NS, Eijkemans MJC, Mannaerts BMJL, Coelingh Bennink HJT, Fauser BCJM. Dynamics of the development of multiple follicles during ovarian stimulation for in vitro fertilization using recombinant follicle-stimulating hormone (Puregon) and various doses of the gonadotropin-releasing hormone antagonist ganirelix (Orgalutran/ Antagon). Fertil Steril 2001;75:688 –93. 8. Albano C, Felberbaum RE, Smitz J, Riethmuller-Winzen H, Engel J, Diedrich K, et al. Ovarian stimulation with HMG: results of a prospective randomized phase III European study comparing the luteinizing hormone-releasing hormone (LHRH)-antagonist cetrorelix and the LHRH-agonist buserelin. Hum Reprod 2000;15:526 –31. 9. Akman MA, Erden HF, Tosun SB, Bayazit N, Aksoy E, Bahceci M. Comparison of agonistic flare-up protocol and antagonist multiple dose protocol in ovarian stimulation of poor responders: results of a prospective randomized trial. Hum Reprod 2001;16:868 –70. 10. Hernandez ER. Embryo implantation and GnRH antagonists. Embryo implantation: the Rubicon for GnRH antagonists. Hum Reprod 2000; 15:1211–6. 11. Kolb BA, Najmabadi S, Paulson RJ. Ultrastructural characteristics of the luteal phase endometrium in patients undergoing controlled ovarian hyperstimulation. Fertil Steril 1997;67:625–30. 12. Kolb BA, Paulson RJ. The luteal phase of cycles utilizing controlled ovarian hyperstimulation and the possible impact of this hyperstimulation on embryo implantation. Am J Obstet Gynecol 1997;67:949 –51. 13. Paulson RJ, Hatch IE, Lobo RA, Sauer MV. Cumulative conception and live birth after oocyte donation: implantations regarding endometrial receptivity. Hum Reprod 1997;12:835–9. 14. Sauer MV, Paulson RJ, Lobo RA. Reversing the natural decline in human fertility. An extended clinical trial of oocyte donation to women of advanced reproductive age. JAMA 1992;268:1275–9. 15. Noyes RW, Herting AT, Rock J. Dating the endometrial biopsy. Fertil Steril 1950;1:3–7. 16. Garcia JE, Acosta AA, Hsiu J-G, Jones HS Jr. Advanced endometrial maturation after ovulation induction with human menopausal gonadotropin/human chorionic gonadotropin for in vitro fertilization. Fertil Steril 1984;41:31–5. 17. Forman RG, Eychenne B, Nessmann C, Frydman R, Robel P. Assessing the early luteal phase in in vitro fertilization cycles: relationships between plasma steroids, endometrial receptors, and endometrial histology. Fertil Steril 1989;51:310 –6. 18. Sundstrom P, Nilsson O, Liedholm P. Scanning electron microscopy of human preimplantation endometrium in normal and clomiphene/ human chorionic gonadotropin-stimulated cycles. Fertil Steril 1983; 40:642–7. 19. Nikas G, Psychoyos A. Uterine pinopods in peri-implantation human endometrium. Clinical relevance. Ann N Y Acad Sci 1997;816:129 –42. 20. Valbuena D, Marin J, de Pablo JL, Remohi J, Pellicer A, Simo´ n C. Increasing levels of estradiol are deleterious to embryonic implantation because they directly affect the embryo. Fertil Steril 2001;76:962–8.
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25. Bustillo M, Stern JJ, Coulam CB. Serum progesterone at the time of human chorionic gonadotrophin does not predict in-vitro fertilization and embryo transfer. Hum Reprod 1995;10:2862–7. 26. Eldestien MC, Seltman HJ, Cox BJ, Robinson SM, Shaw RA, Muasher SJ. Progesterone levels on the day of human chorionic gonadotropin administration in cycles with gonadotropin-releasing hormone agonist suppression are not predictive of pregnancy outcome. Fertil Steril 1990;54:853–7. 27. Legro RS, Ary BA, Paulson RJ, Stanczyk FZ, Sauer MV. Premature luteinization as detected by elevated serum progesterone is associated with a higher pregnancy rate in donor oocyte in-vitro fertilization. Hum Reprod 1993;8:1506 –11.
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