- Email: [email protected]
gonadotropin-releasing hormone antagonist in vitro fertilization cycles during supplementation with progesterone or progesterone and estradiol
The luteal phase of recombinant follicle-stimulating hormone/gonadotropin-releasing hormone antagonist in vitro fertilization cycles during supplementation with progesterone or progesterone and estradiol Human M. Fatemi, M.D., Michel Camus, M.D., Efstratios M. Kolibianakis, M.D., Ph.D., Herman Tournaye, M.D., Ph.D., Evangelos G. Papanikolaou, M.D., Ph.D., Patricio Donoso, M.D., and Paul Devroey, M.D., Ph.D. Centre for Reproductive Medicine, Dutch-Speaking Free University, Brussels, Belgium
Objective: To explore luteal phase hormone profiles in patients stimulated with recombinant FSH and GnRH antagonist for IVF under two different modes of luteal support: P and P with E2. Design: Prospective randomized study. Setting: Patients in an academic reproductive medicine unit. Patient(s): One hundred and three patients undergoing ovarian stimulation with a fixed dose of 200 IU recombinant FSH and GnRH antagonist. Intervention(s): Patients were randomized to receive luteal phase supplementation, either P vaginally (n ⫽ 49) or P and 4 mg E2 orally (n ⫽ 54). Main Outcome Measure(s): Hormonal assessment during the luteal phase on days 1, 4, 7, and 10 after the administration of hCG. Result(s): Hormone levels did not differ during the luteal phase between the two groups with the exception of E2 concentration on day 10 after hCG, which was significantly higher in the E2-supplemented group compared with the P group (median 760 pg/mL, range 2,496 vs. median 589.50 pg/mL, range 2,098). Conclusion(s): Addition of 4 mg E2 for luteal support after stimulation with recombinant FSH and GnRH antagonist does not alter significantly the endocrine profile of the luteal phase until day 7 after hCG. At day 10 after hCG, the E2 levels are significantly higher in the E2-supplemented group. (Fertil Steril威 2007;87:504 – 8. ©2007 by American Society for Reproductive Medicine.) Key Words: Luteal phase support, E2, GnRH antagonists, recombinant FSH
Normal luteal function is essential for establishment and maintenance of pregnancy (1). In stimulated IVF cycles, however, the supraphysiological hormone levels present are associated with a defective luteal phase in almost all patients (2– 4). In an attempt to enhance the probability of pregnancy, different doses, durations, and types of treatments for luteal phase support have been evaluated; however, there is still no agreement regarding the optimal supplementation scheme to be used (5). Supplementation of the luteal phase with P in IVF cycles is the most commonly used method, whereas support with hCG is associated with a higher risk of ovarian hyperstimulation syndrome and its use is limited (1, 6). Controversy still surrounds the value of adding E2 to P (7) to support the luteal phase in agonist cycles (5). In GnRH-antagonist (GnRH-a) cycles, support of the corpora lutea with P is also mandatory after IVF (3, 8); however, no data have been published regarding the therapeutic Received April 21, 2006; revised and accepted July 11, 2006. Reprint requests: Human M. Fatemi, M.D., Vrije Universiteit Brussel/Centrum voor Reproductieve Geneeskunde, Laarbeeklaan 101, 1090 Brussels, Belgium. (Fax: 32-2-4776333; E-mail: [email protected]).
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value of adding E2 to P. Moreover, the effect of such an intervention on the endocrine profile during the luteal phase is not known. The goal of this randomized controlled trial was to explore luteal phase hormone profiles in patients stimulated with recombinant FSH and GnRH-a for IVF with two different modes of luteal support: micronized P and micronized P with the addition of E2 valerate. This study is part of an ongoing trial evaluating the effect of the above manipulation on the probability of pregnancy. MATERIALS AND METHODS Patients Between October 2004 and December 2005, 103 infertile patients were randomized to receive P or P plus E2 for luteal phase support, according to a computer-generated nonconcealed randomization list before initiation of stimulation. The inclusion criteria were age ⱕ39 years; body mass index between 18 and 29; presence of both ovaries; basal levels of E2 (ⱕ80 pg/mL), P (ⱕ1.6 ng/mL), and FSH levels ⬍10 IU/L at initiation of stimulation; and ⬍3 prior IVF cycles. Patients could enter the study only once. The exclusion criteria were presence of polycystic ovarian syndrome diagnosed accord-
Fertility and Sterility姞 Vol. 87, No. 3, March 2007 Copyright ©2007 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/07/$32.00 doi:10.1016/j.fertnstert.2006.07.1521
ing to the revised Rotterdam criteria (Rotterdam European Society of Human Reproduction and Embryology/American Society for Reproductive Medicine, Sponsored Polycystic Ovary Syndrome Consensus Workshop Group 2004) (9), poor responders in a prior cycle (10), presence of endometriosis of American Fertility Society classification stage ⬎2, presence of azoospermia, and the need for preimplantation genetic diagnosis. The research project was approved by our institutional review board. Ovarian Stimulation and IVF/Intracytoplasmic Sperm Injection (ICSI) Procedures The recombinant FSH (Puregon; NV Organon, Oss, the Netherlands) was started in the afternoon of day 2 of the cycle at 200 IU. The dose of recombinant FSH remained unchanged during stimulation until day 10 of the cycle. To inhibit premature LH surge, daily GnRH-a (Orgalutran; 0.25 mg, NV Organon) was used from the morning of day 6 of stimulation. Final oocyte maturation was achieved by administration of 10,000 IU hCG (Pregnyl; NV Organon) as soon as ⱖ3 follicles ⱖ17 mm were present. Oocyte retrieval was carried out 36 hours after hCG administration. Previous studies have described ICSI and IVF procedures in detail (11–14). Embryos were transferred on day 3 after oocyte retrieval. Either one or two embryos were transferred per patient according to the Belgian law on refunding (15). Randomization was performed before initiation of stimulation when the number of cumulus-oocyte complexes retrieved was not available. However, stratification was performed according to the number of embryos transferred. Luteal Supplementation Progesterone Group Luteal phase supplementation with vaginal administration of 600 mg natural micronized P in three separate doses (Utrogestan; Besins-Iscovesco, Paris, France, 100 mg, 2 pills, 3 times daily) was applied starting 1 day after oocyte retrieval and continued until 7 weeks of gestation if pregnancy was achieved.
During the luteal phase, the hormonal assessment was performed on day 1, day 4, day 7, and day 10 after the administration of hCG. Serum hCG tests were performed on days 16 and 18 after the administration of hCG. Serum LH, FSH, hCG, E2, and P were measured with the automated Elecsys immunoanalyzer (Roche Diagnostics, Mannheim, Germany). Intraassay and interassay coefficients of variation were ⬍3% and ⬍4% for LH, ⬍3% and ⬍6% for FSH, ⬍5% and ⬍7% for hCG, ⬍5% and ⬍10% for E2, and ⬍3% and ⬍5% for P, respectively. The upper limit of P level measured was 60 ng/mL. Ultrasound Assessment Ultrasound was performed on day 6 of stimulation and thereafter as necessary to ensure that hCG was injected on the first day that the patient had ⱖ3 follicles ⱖ17 mm. For that purpose, a follicular growth of 2 mm/d was assumed to be present (16). Statistical Analysis This study is part of an ongoing trial evaluating the effect of adding E2 to P in the luteal phase on the probability of pregnancy. The present study reports on the first 103 patients recruited, focusing on their endocrine profile in the luteal phase. Metric variables were analyzed using the independent sample t test, and nonnormally distributed variables were analyzed using the Mann-Whitney U test. Nominal variables were analyzed in the form of frequency tables by using Fisher’s exact test. All tests were two-tailed with a confidence level of 95% (P⬍.05). Metric variables are expressed as mean ⫾ SD. Hormonal levels are expressed as median (range). For assessment of the endocrine profile in the luteal phase, pregnant and nonpregnant patients were considered together because hCG increase in pregnant patients does not occur before day 11 after hCG administration for final oocyte maturation (8, 17).
Progesterone/E2 Group The same treatment as in the previous group was applied with the addition of E2 valerate (Progynova; Schering N.V., Diegem, Belgium) 2 ⫻ 2 mg/d orally also starting 1 day after oocyte retrieval and continued until 7 weeks of gestation if pregnancy was achieved. Progynova 2 mg was administered once in the morning and once in the evening.
RESULTS Patients characteristics and clinical outcome parameters, including indication for treatment, number of oocytes retrieved, number of embryos transferred, number of embryos frozen, units of recombinant FSH administered, and days of stimulation in patients who received the two modes of luteal phase support, were similar as shown in Table 1.
Hormonal Measurements Hormonal assessment during the follicular phase was performed at initiation of stimulation, on day 6, on day 8 of recombinant FSH stimulation, and on the day of hCG administration. Additional blood samples were taken as necessary between antagonist initiation and hCG administration.
The luteal phase endocrine profile (LH, FSH, E2, P) of the two groups compared was similar on day 0, day 1, day 4, and day 7 after hCG administration (Fig. 1). There were two peaks observed for serum E2 in the luteal phase in both groups. The first occurred on day 1 after hCG administration (1,558 pg/mL [range 4,293] in the P group and 1499.50 pg/mL [range 3,815] in the P/E2 group); it was followed by
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TABLE 1 Patient and stimulation characteristics in the P and P/E2 groups. Characteristic Age Body mass index Previous trials Etiology of infertility in the two groups (%) Andrological Endometriosis Idiopathic Tubal Days of stimulation FSH IU Cumulus-oocyte complexes Embryos transferred Embryos frozen at day 3
P
P/E2
P value
32.0 ⫾ 1.6 23.5 ⫾ 13 1.0 ⫾ 7
32.0 ⫾ 1.4 23.0 ⫾ 12 1.0 ⫾ 4
.49 .66 .97 .44
57.1 2.0 16.3 24.5 9.0 ⫾ 7 1,800 ⫾ 1,400 10 ⫾ 36 1.0 ⫾ 1 2.0 ⫾ 14
68.5 2.0 15.0 14.5 9.0 ⫾ 6 1,800 ⫾ 1,200 11 ⫾ 27 1.0 ⫾ 1 2.0 ⫾ 12
.52 .64 .63 .60 .82
Note: Values are means ⫾ SD unless noted. Fatemi. Luteal support: the endocrine profile. Fertil Steril 2007.
a decrease of serum E2 levels until day 4 after hCG (646 pg/mL [range 1,813] in the P group and 692 pg/mL [range 1,926] in the P/E2 group). The second peak of serum E2 occurred on day 7 after hCG (1,249 pg/mL [range 2,909] in the P group and 1,339 pg/mL [range 3,009] in the P/E2 group); it was followed by a decrease in serum E2 levels that resulted in a significantly higher E2 serum concentration on day 10 after hCG administration in the P/E2 group (580.50 pg/mL [range 2,098] in the P group and 760 pg/mL [range 2,496] in the P/E2 group [P⫽.004]). The higher E2 values in this group were accompanied by a marginally lower serum LH (0.30 IU/L [range 3] in the P group and 0.10 IU/L [range 2] in the P/E2 group [P⫽.06]) (Fig. 1). In this study, no patients developed severe ovarian hyperstimulation syndrome. DISCUSSION This is the first study performed in GnRH-a cycles that compares the endocrine profile in the luteal phase under two different supplementation schemes. The study shows that addition of 4 mg E2 to P for luteal support after stimulation with recombinant FSH and GnRH-a does not significantly change the endocrine profile of the luteal phase after GnRH-a recombinant FSH stimulation until day 10 after hCG, when it is associated with a significantly higher serum E2 level. To reduce possible sources of bias in the current study, a fixed dose of recombinant FSH and a fixed protocol of GnRH-a administration were used, whereas the follicular phase ended in the same way in all patients as soon as ⱖ3 follicles ⱖ17 mm were present at ultrasound. Currently, there are no studies that evaluate the role of E2 for luteal support in patients stimulated with GnRH-a. Such 506
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Luteal support: the endocrine profile
an intervention has been evaluated, however, in GnRH agonist cycles with conflicting results. A positive effect of the addition of E2 to P for luteal phase support was confirmed in the studies by Lukaszug et al. (18) and Farhi et al. (19), although not in studies by other investigators (20, 21). The choice of 4 mg E2 supplementation in the current study was arbitrary because no dose-finding studies have been performed thus far regarding this issue in antagonist cycles. Farhi et al. (19), in GnRH agonist cycles, suggested that a beneficial effect of E2 supplementation is present when a dose of 2 mg E2/d was coadministered with progestin support, whereas Smitz et al. (20), in agonist cycles, could not confirm such an effect using a higher E2 dose supplementation (6 mg/d). The results of the current study suggest that addition of 4 mg E2 to P for luteal phase support is not associated with a significant effect in the endocrine profile of the luteal phase, and thus any effect of such an intervention on the probability of pregnancy is expected to be limited. Although an increased E2 level on day 10 after hCG was observed in the group that received 4 mg E2, its importance is not clear. Studies of endometrial histology in the late luteal phase in donor cycles supplemented by the two different modes of luteal support might be necessary to evaluate the importance of such an observation. It should be noted, however, that an effect of higher doses of E2 on the endocrine profile during the luteal phase cannot be excluded. Although the importance of the higher E2 level on day 10 after hCG in the E2 group is unclear, it might be because on day 10 after hCG the E2 produced by the corpora lutea decreases, and thus the effect of adding 4 mg E2 on serum E2 levels becomes evident. The increased serum E2 level in the group that received E2 is accompanied by a marginally lower serum LH level (P⫽.06). If the difference in LH levels Vol. 87, No. 3, March 2007
FIGURE 1 Estradiol, LH, FSH, and P values after hCG administration in the P and in the P/E2 groups.
Fatemi. Luteal support: the endocrine profile. Fertil Steril 2007.
observed represents a true difference between the two groups, it might be attributable to a negative feedback of E2 at the hypothalamus and the pituitary level. In the present study, very low LH levels were present in both groups during the luteal phase, a finding that has been previously reported (22, 23) and probably can be attributed to the high serum E2 levels that are present in the luteal phase after ovarian stimulation for IVF. Because LH is mandatory for the maintenance and normal steroidogenic activity of the human corpus luteum (24), abnormal LH secretion may account for a defective luteal phase (22). Direct administration of E2 into the hypothalamus suppresses LH secretion (25). Estradiol has also been found to reduce in vitro secretion of GnRH from hypothalamic neurons as well as GnRH gene expression in GT1-7 neurons (26). In human men, estrogen was found to have dual site of negative feedback, acting at the hypothalamus to decrease GnRH pulse frequency and at the pituitary to decrease responsiveness to GnRH (27). Moreover, in women with a nonfunctioning hypothalamus, it was shown that the inhibitory effect of E2 on gonadotropin secretion can be reproduced, thereby also providing evidence of a pituitary site of action in women (28). Fertility and Sterility姞
An increase in E2 levels from day 4 to day 7, which was observed in the current study, has also been reported by Beckers et al. (8) and by Dlugi et al. (29). Its explanation is not clear, although it probably signifies a change in hormone production of the corpora lutea, which starts to produce E2 after a first phase of mainly P production. In conclusion, this study shows that the addition of 4 mg E2 to P for luteal support after stimulation with recombinant FSH and GnRH-a does not alter significantly the endocrine profile of the luteal phase after GnRH-a recombinant FSH stimulation until day 7 after hCG. At day 10 after hCG administration, the serum E2 levels are significantly higher in the E2-supplemented group. REFERENCES 1. Penzias AS. Luteal phase support. Fertil Steril 2002;77:318 –23. 2. Ubaldi F, Bourgain C, Tournaye H, Smitz J, van Steirteghem A, Devroey P. Endometrial evaluation by aspiration biopsy on the day of oocyte retrieval in the embryo transfer cycles in patients with serum progesterone rise during the follicular phase. Fertil Steril 1997;67: 521– 6. 3. Kolibianakis EM, Bourgain C, Platteau P, Albano C, Van Steirteghem AC, Devroey P. Abnormal endometrial development occurs during the luteal phase of non-supplemented donor cycles treated with recombi-
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nant follicle-stimulating hormone and gonadotropin-releasing hormone antagonists. Fertil Steril 2003;80:464 – 6. Macklon NS, Fauser BC. Impact of ovarian hyperstimulation on the luteal phase. J Reprod Fertil Suppl 2000;55:101– 8. Pritts E, Atwood A. Luteal phase support in infertility treatment: a meta-analysis of the randomized trials. Hum Reprod 2002;17:2287–99. Papanikolaou EG, Pozzobon C, Kolibianakis EM, Camus M, Tournaye H, Fatemi HM, et al. Incidence and prediction of ovarian hyperstimulation syndrome in women undergoing gonadotropins-releasing hormone antagonist in vitro fertilization cycles. Fertil Steril 2006;85:112–20. Ludwig M, Diedrich K. Evaluation of an optimal luteal phase support protocol in IVF. Acta Obstet Gynecol Scand 2001;80:452– 66. Beckers NG, Macklon NS, Eijkemans MJ, Ludwig M, Felberbaum RF, Diedrich K, et al. Nonsupplemented luteal phase characteristics after the administration of recombinant human chorionic gonadotropins, recombinant luteinizing hormone, or gonadotropins-releasing hormone (GnRh) agonist to induce final oocyte maturation in in vitro fertilization patients after ovarian stimulation with recombinant follicle-stimulating hormone and GnRh antagonist cotreatment. J Clin Endocrinol Metab 2003;88:4186 –92. Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group (2004). Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome. Fertil Steril 2004;81:19 –25. Surrey ES, Schoolcraft WB. Evaluating strategies for improving ovarian response of the poor responder undergoing assisted reproductive techniques. Fertil Steril 2000;73:667–76. Van Steirteghem AC, Nagy Z, Joris H, Liu J, Staessen C, Smitz J, et al. High fertilization and implantation rates after intracytoplasmic sperm injection. Hum Reprod 1993;7:1061– 6. Devroey P, Tjandraprawira K, Mannaerts B, Coelingh-Bennink H, Smitz J, Bonduelle M, et al. A randomized, assessor-blind, groupcomparative efficacy study to compare the effects of Normegon and Metrodin in infertile female patients undergoing in-vitro fertilization. Hum Reprod 1995;2:332–7. Devroey P, Van Steirteghem A. A review of ten years experience of ICSI. Hum Reprod Update 2004;10:19 –28. Van Landuyt L, De Vos A, Joris H, Verheyen G, Devroey P, Van Steirteghem A. Blastocyst formation in in-vitro fertilization versus intracytoplasmic sperm injection cycles: influence of the fertilization procedure. Fertil Steril 2005;83:1397– 403. Moniteur Belge Belgisch staatsblad. 173e Jaargang. 16 June 2003. Pages 32133–57. Kolibianakis EM, Albano C, Camus M, Tournaye H, Van Steirteghem AC, Devroey P. Prolongation of the follicular phase in in-vitro fertilization results in a lower ongoing pregnancy rate in cycles stimulated with recombinant follicle-stimulating hormone and gonadotropin-releasing hormone antagonists. Fertil Steril 2004;82:102–7. Hutchinson-Williams K, Lunenfeld B, Diamond M, Lagy G, Boyers S, De Cherney A. Human chorionic gonadotropin, estradiol, and proges-
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terone profiles in conception and non-conception cycles in an in vitro fertilization program. Fertil Steril 1989;52:441–5. Lukaszuk K, Liss J, Lukaszuk M, Maj B. Optimization of estradiol supplementation during the luteal phase improves the pregnancy rate in women undergoing in vitro fertilization-embryo transfer cycles. Fertil Steril 2005;83:1372– 6. Fahri J, Weissman A, Steinfeld Z, Shorer M, Nahum H, Levran D. Estradiol supplementation during the luteal phase may improve the pregnancy rate in patients undergoing in vitro fertilization-embryo transfer cycles. Fertil Steril 2000;73:761–5. Smitz J, Bourgain C, Van Waesberghe L, Camus M, Devroey P, Van Steirteghem A. A prospective randomized study on estradiol valerate supplementation in addition to intravaginal micronized progesterone in buserelin and HMG induced superovulation. Hum Reprod 1993;8: 40 –5. Lewin A, Benshushan A, Mezker E, Yanai N, Schenker J, Goshen R. The role of estrogen support during the luteal phase of in vitro fertilizationembryo transplant cycles: a comparative study between progesterone alone and estrogen and progesterone support. Fertil Steril 1994;62:212–5. Tavaniotou A, Albano C, Smitz J, Devroey P. Comparison of LH concentrations in the early and mid-luteal phase in IVF cycles after treatment with HMG alone or in association with the GnRh antagonist Cetrorelix. Hum Reprod 2001;16:663–7. Tavaniotou A, Devroey P. Effect of human chorionic gonadotropins on luteal luteinizing hormone concentrations in natural cycles. Fertil Steril 2003;80:654 –5. Casper RF, Yen SS. Induction of luteolysis in the human with a long-acting analog of luteinizing hormone-releasing factor. Science 1979;205:408 –10. Chappel SC, Resko JA, Norman RL, Spies HG. Studies in rhesus monkeys on the site where estrogen inhibits gonadotropins: delivery of 17 beta-estradiol to the hypothalamus and pituitary gland. J Clin Endocrinol Metab 1981;52:1– 8. Roy D, Angelini NL, Belsham DD. Estrogen directly respresses gonadotropin-releasing hormone (GnRh) gene expression in estrogen receptor-alpha (ERalpha) and ERbeta-expressing GTI-7 GnRh neurons. Endocrinology 1999;140:5045–53. Hayes FJ, Seminara SB, Decruz S, Boepple PA, Crowley WF Jr. Aromatase inhibition in the human male reveals a hyphotalamic site of estrogen feedback. J Clin Endocrinol Metab 2000;85:3027–35. Nagahara Y, Miyake A, Tasaka K, Kawamura Y, Aono T, Tanizawa O. Possible site of negative and positive feedback action of estrogen on gonadotropin secretion in normal women. Acta Endocrinol 1985;108: 440 – 4. Dlugi AM, Laufer N, Polan ML, DeCherney AH, Tarlatzis BC, MacLusky NJ, Behrman HR. 17 beta-estradiol and progesterone production by human granulosa-luteal cells isolated from human menopausal gonadotropin-stimulated cycles for in vitro fertilization. J Clin Endocrinol Metab 1984;59:986 –92.
Vol. 87, No. 3, March 2007
Objective: To explore luteal phase hormone profiles in patients stimulated with recombinant FSH and GnRH antagonist for IVF under two different modes of luteal support: P and P with E2. Design: Prospective randomized study. Setting: Patients in an academic reproductive medicine unit. Patient(s): One hundred and three patients undergoing ovarian stimulation with a fixed dose of 200 IU recombinant FSH and GnRH antagonist. Intervention(s): Patients were randomized to receive luteal phase supplementation, either P vaginally (n ⫽ 49) or P and 4 mg E2 orally (n ⫽ 54). Main Outcome Measure(s): Hormonal assessment during the luteal phase on days 1, 4, 7, and 10 after the administration of hCG. Result(s): Hormone levels did not differ during the luteal phase between the two groups with the exception of E2 concentration on day 10 after hCG, which was significantly higher in the E2-supplemented group compared with the P group (median 760 pg/mL, range 2,496 vs. median 589.50 pg/mL, range 2,098). Conclusion(s): Addition of 4 mg E2 for luteal support after stimulation with recombinant FSH and GnRH antagonist does not alter significantly the endocrine profile of the luteal phase until day 7 after hCG. At day 10 after hCG, the E2 levels are significantly higher in the E2-supplemented group. (Fertil Steril威 2007;87:504 – 8. ©2007 by American Society for Reproductive Medicine.) Key Words: Luteal phase support, E2, GnRH antagonists, recombinant FSH
Normal luteal function is essential for establishment and maintenance of pregnancy (1). In stimulated IVF cycles, however, the supraphysiological hormone levels present are associated with a defective luteal phase in almost all patients (2– 4). In an attempt to enhance the probability of pregnancy, different doses, durations, and types of treatments for luteal phase support have been evaluated; however, there is still no agreement regarding the optimal supplementation scheme to be used (5). Supplementation of the luteal phase with P in IVF cycles is the most commonly used method, whereas support with hCG is associated with a higher risk of ovarian hyperstimulation syndrome and its use is limited (1, 6). Controversy still surrounds the value of adding E2 to P (7) to support the luteal phase in agonist cycles (5). In GnRH-antagonist (GnRH-a) cycles, support of the corpora lutea with P is also mandatory after IVF (3, 8); however, no data have been published regarding the therapeutic Received April 21, 2006; revised and accepted July 11, 2006. Reprint requests: Human M. Fatemi, M.D., Vrije Universiteit Brussel/Centrum voor Reproductieve Geneeskunde, Laarbeeklaan 101, 1090 Brussels, Belgium. (Fax: 32-2-4776333; E-mail: [email protected]).
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value of adding E2 to P. Moreover, the effect of such an intervention on the endocrine profile during the luteal phase is not known. The goal of this randomized controlled trial was to explore luteal phase hormone profiles in patients stimulated with recombinant FSH and GnRH-a for IVF with two different modes of luteal support: micronized P and micronized P with the addition of E2 valerate. This study is part of an ongoing trial evaluating the effect of the above manipulation on the probability of pregnancy. MATERIALS AND METHODS Patients Between October 2004 and December 2005, 103 infertile patients were randomized to receive P or P plus E2 for luteal phase support, according to a computer-generated nonconcealed randomization list before initiation of stimulation. The inclusion criteria were age ⱕ39 years; body mass index between 18 and 29; presence of both ovaries; basal levels of E2 (ⱕ80 pg/mL), P (ⱕ1.6 ng/mL), and FSH levels ⬍10 IU/L at initiation of stimulation; and ⬍3 prior IVF cycles. Patients could enter the study only once. The exclusion criteria were presence of polycystic ovarian syndrome diagnosed accord-
Fertility and Sterility姞 Vol. 87, No. 3, March 2007 Copyright ©2007 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/07/$32.00 doi:10.1016/j.fertnstert.2006.07.1521
ing to the revised Rotterdam criteria (Rotterdam European Society of Human Reproduction and Embryology/American Society for Reproductive Medicine, Sponsored Polycystic Ovary Syndrome Consensus Workshop Group 2004) (9), poor responders in a prior cycle (10), presence of endometriosis of American Fertility Society classification stage ⬎2, presence of azoospermia, and the need for preimplantation genetic diagnosis. The research project was approved by our institutional review board. Ovarian Stimulation and IVF/Intracytoplasmic Sperm Injection (ICSI) Procedures The recombinant FSH (Puregon; NV Organon, Oss, the Netherlands) was started in the afternoon of day 2 of the cycle at 200 IU. The dose of recombinant FSH remained unchanged during stimulation until day 10 of the cycle. To inhibit premature LH surge, daily GnRH-a (Orgalutran; 0.25 mg, NV Organon) was used from the morning of day 6 of stimulation. Final oocyte maturation was achieved by administration of 10,000 IU hCG (Pregnyl; NV Organon) as soon as ⱖ3 follicles ⱖ17 mm were present. Oocyte retrieval was carried out 36 hours after hCG administration. Previous studies have described ICSI and IVF procedures in detail (11–14). Embryos were transferred on day 3 after oocyte retrieval. Either one or two embryos were transferred per patient according to the Belgian law on refunding (15). Randomization was performed before initiation of stimulation when the number of cumulus-oocyte complexes retrieved was not available. However, stratification was performed according to the number of embryos transferred. Luteal Supplementation Progesterone Group Luteal phase supplementation with vaginal administration of 600 mg natural micronized P in three separate doses (Utrogestan; Besins-Iscovesco, Paris, France, 100 mg, 2 pills, 3 times daily) was applied starting 1 day after oocyte retrieval and continued until 7 weeks of gestation if pregnancy was achieved.
During the luteal phase, the hormonal assessment was performed on day 1, day 4, day 7, and day 10 after the administration of hCG. Serum hCG tests were performed on days 16 and 18 after the administration of hCG. Serum LH, FSH, hCG, E2, and P were measured with the automated Elecsys immunoanalyzer (Roche Diagnostics, Mannheim, Germany). Intraassay and interassay coefficients of variation were ⬍3% and ⬍4% for LH, ⬍3% and ⬍6% for FSH, ⬍5% and ⬍7% for hCG, ⬍5% and ⬍10% for E2, and ⬍3% and ⬍5% for P, respectively. The upper limit of P level measured was 60 ng/mL. Ultrasound Assessment Ultrasound was performed on day 6 of stimulation and thereafter as necessary to ensure that hCG was injected on the first day that the patient had ⱖ3 follicles ⱖ17 mm. For that purpose, a follicular growth of 2 mm/d was assumed to be present (16). Statistical Analysis This study is part of an ongoing trial evaluating the effect of adding E2 to P in the luteal phase on the probability of pregnancy. The present study reports on the first 103 patients recruited, focusing on their endocrine profile in the luteal phase. Metric variables were analyzed using the independent sample t test, and nonnormally distributed variables were analyzed using the Mann-Whitney U test. Nominal variables were analyzed in the form of frequency tables by using Fisher’s exact test. All tests were two-tailed with a confidence level of 95% (P⬍.05). Metric variables are expressed as mean ⫾ SD. Hormonal levels are expressed as median (range). For assessment of the endocrine profile in the luteal phase, pregnant and nonpregnant patients were considered together because hCG increase in pregnant patients does not occur before day 11 after hCG administration for final oocyte maturation (8, 17).
Progesterone/E2 Group The same treatment as in the previous group was applied with the addition of E2 valerate (Progynova; Schering N.V., Diegem, Belgium) 2 ⫻ 2 mg/d orally also starting 1 day after oocyte retrieval and continued until 7 weeks of gestation if pregnancy was achieved. Progynova 2 mg was administered once in the morning and once in the evening.
RESULTS Patients characteristics and clinical outcome parameters, including indication for treatment, number of oocytes retrieved, number of embryos transferred, number of embryos frozen, units of recombinant FSH administered, and days of stimulation in patients who received the two modes of luteal phase support, were similar as shown in Table 1.
Hormonal Measurements Hormonal assessment during the follicular phase was performed at initiation of stimulation, on day 6, on day 8 of recombinant FSH stimulation, and on the day of hCG administration. Additional blood samples were taken as necessary between antagonist initiation and hCG administration.
The luteal phase endocrine profile (LH, FSH, E2, P) of the two groups compared was similar on day 0, day 1, day 4, and day 7 after hCG administration (Fig. 1). There were two peaks observed for serum E2 in the luteal phase in both groups. The first occurred on day 1 after hCG administration (1,558 pg/mL [range 4,293] in the P group and 1499.50 pg/mL [range 3,815] in the P/E2 group); it was followed by
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TABLE 1 Patient and stimulation characteristics in the P and P/E2 groups. Characteristic Age Body mass index Previous trials Etiology of infertility in the two groups (%) Andrological Endometriosis Idiopathic Tubal Days of stimulation FSH IU Cumulus-oocyte complexes Embryos transferred Embryos frozen at day 3
P
P/E2
P value
32.0 ⫾ 1.6 23.5 ⫾ 13 1.0 ⫾ 7
32.0 ⫾ 1.4 23.0 ⫾ 12 1.0 ⫾ 4
.49 .66 .97 .44
57.1 2.0 16.3 24.5 9.0 ⫾ 7 1,800 ⫾ 1,400 10 ⫾ 36 1.0 ⫾ 1 2.0 ⫾ 14
68.5 2.0 15.0 14.5 9.0 ⫾ 6 1,800 ⫾ 1,200 11 ⫾ 27 1.0 ⫾ 1 2.0 ⫾ 12
.52 .64 .63 .60 .82
Note: Values are means ⫾ SD unless noted. Fatemi. Luteal support: the endocrine profile. Fertil Steril 2007.
a decrease of serum E2 levels until day 4 after hCG (646 pg/mL [range 1,813] in the P group and 692 pg/mL [range 1,926] in the P/E2 group). The second peak of serum E2 occurred on day 7 after hCG (1,249 pg/mL [range 2,909] in the P group and 1,339 pg/mL [range 3,009] in the P/E2 group); it was followed by a decrease in serum E2 levels that resulted in a significantly higher E2 serum concentration on day 10 after hCG administration in the P/E2 group (580.50 pg/mL [range 2,098] in the P group and 760 pg/mL [range 2,496] in the P/E2 group [P⫽.004]). The higher E2 values in this group were accompanied by a marginally lower serum LH (0.30 IU/L [range 3] in the P group and 0.10 IU/L [range 2] in the P/E2 group [P⫽.06]) (Fig. 1). In this study, no patients developed severe ovarian hyperstimulation syndrome. DISCUSSION This is the first study performed in GnRH-a cycles that compares the endocrine profile in the luteal phase under two different supplementation schemes. The study shows that addition of 4 mg E2 to P for luteal support after stimulation with recombinant FSH and GnRH-a does not significantly change the endocrine profile of the luteal phase after GnRH-a recombinant FSH stimulation until day 10 after hCG, when it is associated with a significantly higher serum E2 level. To reduce possible sources of bias in the current study, a fixed dose of recombinant FSH and a fixed protocol of GnRH-a administration were used, whereas the follicular phase ended in the same way in all patients as soon as ⱖ3 follicles ⱖ17 mm were present at ultrasound. Currently, there are no studies that evaluate the role of E2 for luteal support in patients stimulated with GnRH-a. Such 506
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an intervention has been evaluated, however, in GnRH agonist cycles with conflicting results. A positive effect of the addition of E2 to P for luteal phase support was confirmed in the studies by Lukaszug et al. (18) and Farhi et al. (19), although not in studies by other investigators (20, 21). The choice of 4 mg E2 supplementation in the current study was arbitrary because no dose-finding studies have been performed thus far regarding this issue in antagonist cycles. Farhi et al. (19), in GnRH agonist cycles, suggested that a beneficial effect of E2 supplementation is present when a dose of 2 mg E2/d was coadministered with progestin support, whereas Smitz et al. (20), in agonist cycles, could not confirm such an effect using a higher E2 dose supplementation (6 mg/d). The results of the current study suggest that addition of 4 mg E2 to P for luteal phase support is not associated with a significant effect in the endocrine profile of the luteal phase, and thus any effect of such an intervention on the probability of pregnancy is expected to be limited. Although an increased E2 level on day 10 after hCG was observed in the group that received 4 mg E2, its importance is not clear. Studies of endometrial histology in the late luteal phase in donor cycles supplemented by the two different modes of luteal support might be necessary to evaluate the importance of such an observation. It should be noted, however, that an effect of higher doses of E2 on the endocrine profile during the luteal phase cannot be excluded. Although the importance of the higher E2 level on day 10 after hCG in the E2 group is unclear, it might be because on day 10 after hCG the E2 produced by the corpora lutea decreases, and thus the effect of adding 4 mg E2 on serum E2 levels becomes evident. The increased serum E2 level in the group that received E2 is accompanied by a marginally lower serum LH level (P⫽.06). If the difference in LH levels Vol. 87, No. 3, March 2007
FIGURE 1 Estradiol, LH, FSH, and P values after hCG administration in the P and in the P/E2 groups.
Fatemi. Luteal support: the endocrine profile. Fertil Steril 2007.
observed represents a true difference between the two groups, it might be attributable to a negative feedback of E2 at the hypothalamus and the pituitary level. In the present study, very low LH levels were present in both groups during the luteal phase, a finding that has been previously reported (22, 23) and probably can be attributed to the high serum E2 levels that are present in the luteal phase after ovarian stimulation for IVF. Because LH is mandatory for the maintenance and normal steroidogenic activity of the human corpus luteum (24), abnormal LH secretion may account for a defective luteal phase (22). Direct administration of E2 into the hypothalamus suppresses LH secretion (25). Estradiol has also been found to reduce in vitro secretion of GnRH from hypothalamic neurons as well as GnRH gene expression in GT1-7 neurons (26). In human men, estrogen was found to have dual site of negative feedback, acting at the hypothalamus to decrease GnRH pulse frequency and at the pituitary to decrease responsiveness to GnRH (27). Moreover, in women with a nonfunctioning hypothalamus, it was shown that the inhibitory effect of E2 on gonadotropin secretion can be reproduced, thereby also providing evidence of a pituitary site of action in women (28). Fertility and Sterility姞
An increase in E2 levels from day 4 to day 7, which was observed in the current study, has also been reported by Beckers et al. (8) and by Dlugi et al. (29). Its explanation is not clear, although it probably signifies a change in hormone production of the corpora lutea, which starts to produce E2 after a first phase of mainly P production. In conclusion, this study shows that the addition of 4 mg E2 to P for luteal support after stimulation with recombinant FSH and GnRH-a does not alter significantly the endocrine profile of the luteal phase after GnRH-a recombinant FSH stimulation until day 7 after hCG. At day 10 after hCG administration, the serum E2 levels are significantly higher in the E2-supplemented group. REFERENCES 1. Penzias AS. Luteal phase support. Fertil Steril 2002;77:318 –23. 2. Ubaldi F, Bourgain C, Tournaye H, Smitz J, van Steirteghem A, Devroey P. Endometrial evaluation by aspiration biopsy on the day of oocyte retrieval in the embryo transfer cycles in patients with serum progesterone rise during the follicular phase. Fertil Steril 1997;67: 521– 6. 3. Kolibianakis EM, Bourgain C, Platteau P, Albano C, Van Steirteghem AC, Devroey P. Abnormal endometrial development occurs during the luteal phase of non-supplemented donor cycles treated with recombi-
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