RBMOnline - Vol 18 No 1. 2009 67-72 Reproductive BioMedicine Online; www.rbmonline.com/article/3551 on web 14 November 2008 Article Frozen thawed cleavage-stage embryo transfer cycles after previous GnRH agonist or antagonist stimulation Professor Mustafa Bahçeci completed his Obstetrics and Gynaecology residency in Ankara University, Turkey in 1984 where he received his MD degree. He continued on the Faculty and helped to develop the reproductive endocrinology department. He received postgraduate training in reproductive endocrinology and endoscopic surgery in USA. He returned to Istanbul and founded the German Hospital IVF unit in 1996, which is now one of the largest centres in Turkey, treating more than 2000 cases a year. One of his major interests is the treatment of advanced endometriosis with endoscopic surgery. Dr Professor Mustafa Bahçeci Mustafa Bahçeci 1, Ulun Ulug, Halit Fırat Erden, Suleyman Tosun, Nadir Çiray Bahçeci Women Health Care Centre and German Hospital in Istanbul, Istanbul, Turkey 1 Correspondence: e-mail: mbahceci@superonline.com Abstract This study presents the results of fresh and frozen thawed embryo transfers in women undergoing IVF with gonadotrophinreleasing hormone (GnRH) agonists and GnRH antagonists. By evaluating cycle outcomes, the impact of two different protocols on the endometrium was indirectly evaluated. For 714 women, embryos were frozen following day-3 fresh embryo transfer and the outcome of those fresh cycles (329 agonist cycles and 290 antagonist cycles) and subsequent frozen thawed embryo transfer (91 agonist cycles and 104 antagonist cycles) were evaluated. Peak oestradiol concentrations of both groups were similar; however, significantly more oocytes were retrieved and more embryos frozen in the agonist versus antagonist group (both P = 0.0001). In fresh embryo transfer cycles, implantation and pregnancy rates in the agonist versus antagonist group were 42.3% versus 32.0% (P = 0.0001) and 68.6% versus 58.2% (P = 0.009) respectively. However, neither implantation or pregnancy rate significantly differed among frozen thawed embryo transfer cycles between the two groups (21.4% versus 23.5% and 52.2% versus 52.4% respectively). These results suggest that ovarian stimulation parameter outcomes of GnRH antagonist cycles were not inferior to GnRH agonist cycles, therefore reduced embryo implantation and pregnancy rates in GnRH antagonist cycles can be attributable to possible deleterious effects on the endometrium. Keywords: embryo, endometrium, frozen, GnRH antagonist Introduction During the early years of IVF, it became apparent that premature LH surge could complicate ovarian stimulation and decrease pregnancy rates (Loumaye, 1990). Pituitary downregulation using gonadotrophin-releasing hormone (GnRH) agonists decreased the proportion of cycle cancellation due to premature LH surge from approximately 20% to 2% (Loumaye, 1990) and led to a significant improvement of IVF outcome (Hughes et al., 1992). On the other hand, agonist usage was accompanied by ovarian cyst occurrence, oestrogen deprivation symptoms, an increased consumption of gonadotrophins and lack of immediate pituitary responsiveness following agonist discontinuation (Smitz et al., 1992). More recently introduced, GnRH antagonists allowed immediate suppression of premature LH surge in the mid-follicular phase offering a rational way to perform ovarian stimulation. The confirmed advantages of GnRH antagonist over agonists were decreased amount of gonadotrophin utilization and a shorter period of stimulation. However, the expectation that pregnancy rates with antagonists would be at least equal to those achieved by GnRH agonists was not realised (Al-Inany et al., 2007). Although several confounding variables, such as learning curve of such a new protocol among physicians or tendency of selecting poor-responder patients in clinical trials, have been taken into consideration for the relatively reduced pregnancy rates in women in whom GnRH antagonists were utilized. A recent analysis also showed that more patients over 35 years old underwent assisted reproduction treatment with GnRH antagonists than GnRH agonists in Germany (Griesinger et al., 67 2009 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB23 8DB, UK
68 2005). Therefore it appears that clinical experience in terms of IVF outcome mostly derived from the results of women with relatively poor prognosis. In this regard, it is proposed that those cycles ending with transfer of good-quality embryos and remaining with goodquality cleavage-stage embryos to be frozen should be defined as good prognosis. In addition, evaluation of the following frozen thawed embryo transfer cycles of those cases will eventually present the outcome by encompassing endometrium as a confounding variable in the previous fresh cycle. Therefore, this study has evaluated and compared outcome of fresh cycles of women undergoing IVF with GnRH agonists and GnRH antagonists and frozen thawed embryo transfer cycles of those women. Materials and methods Patients This study retrospectively analysed the outcome in women who had remaining good-quality embryos frozen following fresh embryo transfer between January 2004 and December 2005. Out of 5889 cycles, 714 women (12.1%) had embryos frozen following a single day-3 fresh embryo transfer during this period. After exclusion of frozen embryos generated from cancelled fresh cycles (prevention from ovarian hyperstimulation syndrome, or couple s request) and those in whom surgically retrieved spermatozoa used, 619 patients remained. GnRH agonist for ovarian stimulation was utilized in 329 women and GnRH antagonist in 290 women. Subsequently 195 women underwent frozen thawed embryo transfer among those individuals (91 from the agonist group and 104 from the antagonist group) during the 2-year period. All patients underwent a single frozen thawed embryo transfer cycle. Ovarian stimulation with GnRH agonist The ovulation induction protocol for ovarian stimulation began with pituitary desensitization by GnRH agonist (Lucrin; Abbott, France) in the mid-luteal phase of the preceding menstrual period. Administration of gonadotrophins (Gonal F; Serono, Switzerland; or Menogon; Ferring, Sweden) was initiated when serum oestradiol concentration fell below 50 pg/ ml. The starting regimen was fixed for the first 4 days (150 300 IU) and subsequently adjusted according to the individual ovarian response. Ovarian stimulation for GnRH antagonist Gonadotrophins (Gonal F), at dosages of 150 300 IU, were started on day 2, and, when the leading follicle reached 14 mm in diameter, 0.25 mg of cetrorelix (Cetrotide ; Serono) was administered daily until the day of human chorionic gonadotrophin (HCG) injection. The starting regimen was fixed for the first 4 days, and thereafter the gonadotrophin dose was adjusted according to the individual ovarian response. Human menopausal gonadotrophin (Menogon, Ferring, Sweden), at a dose of 75 IU per day, was also administered starting the same day as antagonist. In both protocols when at least two follicles reached 18 mm in diameter, HCG (Pregnyl ; Organon) 10,000 IU i.m. was administered. Oocytes were retrieved 35 h after HCG injection. The method for assisted fertilization in the study centre was intracytoplasmic sperm injection, which has been used in all patients regardless of infertility aetiology. Three days after oocyte retrieval, the embryos were transferred transcervically under ultrasound guidance. The luteal phase was supported by 100 mg/day i.m. progesterone in oil. Endometrial preparation for frozen thawed embryo transfer Oestradiol as a transdermal therapeutic system 2 mg (Estraderm TTS; Novartis, Istanbul, Turkey) was initiated on cycle day 1 and the dose was gradually increased every 2 days. Progesterone in oil 50 mg was added by cycle day 15 and embryos were transferred on cycle day 18. Embryo selection and freezing and thawing processes Institutional criteria for cryopreservation of embryos on day 3 were as follows. Embryos displaying six or more even or uneven blastomeres with 20% fragmentation and without multinucleation. For the cryopreservation procedure, the embryos were placed in a four-well Petri dish, which was placed in a laminar flow cabinet at room temperature. All solutions were purchased from SAGE-Biopharma (USA). Embryos were washed three to four times in 0.7 ml Cryo Kit Diluent solution and were immersed into 0.7 ml 1.5 mol/l propanediol for 10 min. Embryos were then washed in 0.7 ml solution of 1.5 mol/l propanediol and 0.1 mol/l sucrose and were immediately placed in a vial containing the same solution. Cryopreservation procedure continued under a controlled programme (Planer Kryo 10, Series III) and seeding was performed manually. Thawing procedure was performed at room temperature in a four-well Petri dish, which was placed in a laminar flow cabinet. All solutions were purchased from SAGE-Biopharma (USA) and were brought to room temperature before thawing procedure initiated. Vials containing the embryos were taken out of the liquid nitrogen tanks and were immersed in a water bath at 30 C until thawing (approximately 1 min). The vial was then kept in room temperature for 5 min. The solution in the vial was poured into a dish and the embryos were taken into a pipette to be transferred into 0.7 ml of 0.5 mol/l sucrose. Embryos were immersed in this solution for 10 min and were taken into 0.7 ml of 0.2 mol/l sucrose for incubation for another 10 min. Then they were washed and immersed in 0.7 ml Cryo Kit Diluent solution for 5 min. Embryos were subsequently placed in Quinn s Cleavage Medium (SAGE-Biopharma, USA) and were incubated until transfer. Clinical pregnancy was defined when a transvaginal ultrasound scan performed 3 4 weeks after embryo transfer revealed a gestational sac together with a yolk sac. Student s t-test and the Mann Whitney U-test were utilized for continuous variables and chi-squared test was used for categorical variables.
Results Both fresh embryo transfer groups (agonist and antagonist) demonstrated similar characteristics in terms of age, baseline serum gonadotrophin concentrations, duration of infertility, number of previous assisted cycles and underlying infertility aetiology (Table 1). The ovarian stimulation and fresh embryo transfer cycle characteristics and outcomes of both groups are depicted in Table 2. The amount of LH content of gonadotrophin, oestradiol concentration on HCG day and number of embryos transferred were not different between fresh embryo transfer groups. The duration of stimulation with gonadotrophins was significantly shorter in antagonist group than agonist group (P = 0.0002) while the total amount of gonadotrophin consumed per patient was significantly reduced in agonist group (P = 0.0003). While the total number of oocytes retrieved was significantly higher in the antagonist versus the agonist group (P = 0.0001), metaphase II ratio was significantly lower in the antagonist group (P = 0.003). In the agonist group, more cycles had undergone coasting during ovarian stimulation. In the antagonist group, more embryos were frozen compared with agonist group. However, implantation and pregnancy rates were significantly higher in the agonist compared with the antagonist group (P = 0.0001 and P = 0.009 respectively). Among the evaluated frozen thawed embryo transfer cycles, 19 and 13 patients out of agonist and antagonist groups, respectively, had conceived in their previous fresh embryo transfer (20% and 12.5%, not significantly different). The outcomes of frozen thawed embryo transfer cycles are shown in Table 3. Among the frozen thawed embryo transfer Table 1. Characteristics of two groups of women undergoing ovarian stimulation with gonadotrophin-releasing hormone agonist or antagonist. Agonist (n = 329) Antagonist (n = 290) Mean age (years) 30.5 ± 4.4 (19 42) 31.0 ± 5.3 (18 43) Baseline FSH (IU/l) 6.0 ± 2.0 6.4 ± 2.4 Baseline LH (IU/l) 6.3 ± 3.9 6.9 ± 4.7 Duration of infertility (years) 7.9 ± 4.6 (1 19) 7.6 ± 4.8 (1 20) No. of previous trials 1.6 ± 0.4 (0 3) 1.2 ± 0.5 (0 3) Aetiology Female (%) 37.6 33.0 Male (%) 40.7 43.1 Unexplained (%) 13.1 14.5 Combined (%) 8.5 9.2 Values are mean ± SD or mean ± SD (range) unless otherwise stated. There were no statistically significant differences between the two groups. Table 2. Comparison of cycle characteristics and outcomes in women undergoing ovarian stimulation and IVF by intracytoplasmic sperm injection and fresh embryo transfer in gonadotrophin-releasing hormone agonist or antagonist cycles. Agonist (n = 329) Antagonist (n = 290) P-value Duration of gonadotrophin 11.7 ± 1.5 11.2 ± 2.1 0.0002 stimulation (days) No. of gonadotrophin ampoules 29.8 ± 11.8 33.2 ± 12.5 0.0003 consumed (75 IU each) LH content (%) 15.2 16.1 NS Oestradiol on HCG day (pg/ml) 3388.5 ± 1073.0 3530.8 ± 1155.1 NS No. of coasted cycles (%) 122 (37.1) 13 (4.5) 0.0001 (OR 7.3, 95% CI 7.3 13.4) No. of oocytes retrieved 18.5 ± 6.1 23.0 ± 10.0 0.0001 MII/total no. of oocytes (%) 5215/6086 (85.7) 5590/6670 (83.8) 0.003 (OR 1.1, 95% CI 1.0 1.2) No. of frozen embryos 6.8 ± 2.6 8.0 ± 3.9 0.0001 No. of embryos transferred 2.7 ± 0.5 2.6 ± 0.5 NS Implantation rate (%) 378/893 (42.3) 250/779 (32.1) 0.0001 (OR 1.5, 95% CI 1.2 1.9) No. of clinical pregnancies (%) 226 (68.7) 169 (58.3) 0.009 (OR 1.9, 95% CI 1.1 2.1) No. of multiple pregnancies (%) 109 (48.2) 80 (47.3) NS No. of miscarriages before 21 (9.3) 15 (8.9) NS 10 weeks (%) Values are mean ± SD unless otherwise stated. HCG = human chorionic gonadotrophin; MII = metaphase II; NS = not statistically significant. 69
Table 3. Comparison of frozen thawed embryo transfer cycles in two groups of women who had previously undergone ovarian stimulation with either gonadotrophin-releasing hormone agonist or antagonist. Agonist Antagonist No. of thawing procedures 91 104 No. of transfer cycles 90 103 Endometrial thickness (mm) 9.8 ± 1.3 9.6 ± 1.5 Oestradiol on embryo transfer day (pg/ml) 348.7 ± 200.8 349.1 ± 212.3 No. of embryos transferred 3.1 ± 0.5 3.1 ± 0.8 No. of clinical pregnancies 47 54 Implantation rate (%) 58/270 (21.5) 69/293 (23.5) Clinical pregnancy rate/embryo transfer cycle (%) 52.2 52.4 No. of multiple pregnancies (%) 10 (21.3) 14 (26.0) No. of miscarriages before 10 weeks (%) 6 (12.8) 5 (9.3) Values are mean ± SD unless otherwise stated. There were no statistically significant differences between the two groups. 70 cycles, both groups had similar characteristics in terms of endometrial thickness and oestradiol concentration on transfer day and mean number of embryos transferred; thus neither implantation nor pregnancy rates were significantly different. Discussion This study examined the effectiveness of different pituitary desensitization methods for ovarian stimulation among women with good ovarian responses. Therefore, common criticisms of previous studies comparing GnRH agonist/antagonist cycles in relatively poor-responder patients were avoided by using this design. However, the findings of reduced implantation and pregnancy rates in fresh GnRH antagonist cycles compared with agonist cycles are in accordance with a recently published Cochrane review (Al-Inany et al., 2007). By evaluating outcome in frozen thawed embryo transfer cycles generated via both agonist and antagonist protocols, the effects of two different protocols on the endometrium of women undergoing assisted reproduction treatment has been indirectly evaluated. The centre s freezing protocol also permitted the selection of good-quality embryos in both groups, rendering them comparable in terms of embryo yield following ovulation induction in index cycles. Demonstrating lower pregnancy rates among GnRH antagonist patients in fresh embryo transfer cycles and similar pregnancy rates in frozen thawed embryo transfer compared with GnRH agonist patients leads to the suggestion that a potentially deleterious effect of GnRH antagonists on the endometrium may explain some of the difference observed in outcomes. Given the fact that more oocytes and more embryos were frozen following ovarian stimulation in the antagonist group, ovarian responses among those individuals were not inferior to those seen in agonist-stimulated cases. Outcomes of frozen thawed embryo transfer originating from GnRH antagonist cycles have been addressed in several studies. In contrast to the present study, these studies evaluated effects of antagonists on day-2 embryo and blastocyst transfers. Previous investigators concluded that neither the duration nor the dosage of the GnRH antagonist employed affected the probability of blastocyst replacement or of achieving pregnancy in subsequent frozen thawed cycles (Kol et al., 1999; Zikopoulos et al., 2004). It has been shown that implantation was independent of the type of gonadotrophin-releasing hormone analogue chosen for the collection cycle when previously cryopreserved embryos were used (Seelig et al., 2002; Medved et al., 2006; Eldar-Geva et al., 2007). In accordance with the results presented here, the authors concluded that lower IVF embryo transfer success rates using GnRH antagonist/gnrh agonist protocols do not appear to be related to an adverse effect on oocyte quality. Receptors for GnRH have been found in the ovary and the endometrium (Gründker et al., 2002), prompting suggestions that GnRH antagonists may have extra-pituitary effects (Hernandez, 2000). It has been shown that GnRH antagonists competitively and selectively bind to GnRH receptors, and in-vitro studies of receptor binding profiles among GnRH antagonists have shown differential effects. Cetrorelix, for example, exerts a binding affinity about 20 times higher than native GnRH (Hernandez, 2000). Ovarian paracrine function has also been assessed in prior studies. Cunha-Filho et al. (2005) demonstrated that GnRH antagonist administration in infertile patients undergoing IVF did not alter the follicular fluid concentrations of vascular endothelial growth factor (VEGF) and inhibin A, nor did it alter the apparent maturation capacity and quality of treated oocytes. Furthermore, in a recent analysis, no difference was found in the follicular dynamics of paracrine factors such as the inhibins, insulin-like growth factor-1 or epidermal growth factor in women undergoing ovarian stimulation with either agonists or antagonists (Ulug et al., 2007). The expression of various growth factors and their receptors in the uterus in a temporal and cell-specific manner during the preimplantation period suggests that some of these factors are important for implantation (Guzeloglu-Kayisli et al., 2007). The impact of GnRH antagonists on endometrium have been studied in several reports. Human endometrium expresses GnRH receptors, and GnRH analogues alter the expression of transforming growth factor- ), fibronectin and antagonist analogues increase L-selectin in endometrial cells (Luo et al.,
2004; Vlahos et al., 2006) that are related to the implantation process. Furthermore, it was shown that, cetrorelix inhibits the regulatory effects of GnRH on plasminogen activator inhibitor-1 expression in decidual stromal cells (Chou et al., 2003a). Matrix metalloproteinases (MMP) and their endogenous inhibitors, tissue-specific inhibitors of matrix metalloproteinases (TIMP), play key roles in the human endometrium in preparation for pregnancy. GnRH increases MMP-2 and MMP-9 mrna concentrations in endometrial cell cultures (Chou et al., 2002). On the other hand, a GnRH antagonist, antide, was capable of inhibiting GnRH-mediated increases in concentrations of MMP-2 and MMP-9 (Chou et al., 2003b). Thus, Raga et al. (1999) proposed that GnRH may play an important role in placental tissue organization and in the early embryonic maternal placental dialogue by enhancing trophoblast invasion through the specific inhibition of TIMP. It was also demonstrated that ovarian stimulation with GnRH agonists, but not GnRH antagonists, could restore physiological endometrial secretions and improve uterine receptivity in mice (Ruan et al., 2006). Vani et al. (2007) stated that changes in the expression of sex-steroid receptors and metabolizing enzymes may lead to alterations in the activity and intracellular availability of oestrogens, progestins and androgens in the endometrium of women treated with the GnRH antagonist, cetrorelix. On the other hand, GnRH agonists, but not antagonists, appeared to exert direct effects in endometrial cell cultures. Specifically, they enhanced the percentage of apoptotic cells, increased the expression of pro-apoptotic proteins (Bax and FasL) and decreased the release of pro-mitogenic cytokines (interleukin- 1 and VEGF) and anti-apoptotic proteins (Bcl-2) (Meresman et al., 2003; Bilotas et al., 2007). Several studies have evaluated the impact of GnRH antagonist treatment on endometrial histology but have reported conflicting findings. Stimulation of endometrial maturation at the time of oocyte retrieval was observed in a group of women treated with single-dose cetrorelix (Kolibianakis et al., 2002). In contrast, it was found that GnRH antagonists had no effect on the endometrium of regularly menstruating women as assessed by either histological dating or morphometric analysis (Sirayapiwat et al., 2007) and no differences in endometrial growth patterns or luteal-phase endometrial pathology between patients treated with either GnRH agonists or antagonists (Saadat et al., 2004). No relevant alterations were also observed in endometrial development during early and mid-luteal phases in women undergoing ovarian stimulation for oocyte donation following daily treatment with either a standard- or high-dose GnRH antagonist (Simon et al., 2005). It appears, therefore, that administration of GnRH antagonists results in changes in the endometrium, which have been demonstrated at the subcellular level, but not observed histologically. The present study was retrospective; hence the conclusions derived from it should be interpreted with caution. Nevertheless, these results suggest that use of GnRH antagonists results in efficient embryo yield, with consistent early treatment outcomes. The inferior outcomes seen in antagonist-treated patients compared with agonist-treated patients probably occur later, consistent with deleterious endometrial effects due to GnRH antagonists. References Al-Inany HG, Abou-Setta AM, Aboulghar M 2007 Gonadotrophinreleasing hormone antagonists for assisted conception: a Cochrane review. Reproductive BioMedicine Online 14, 640 649. Bilotas M, Barañao RI, Buquet R et al. 2007 Effect of GnRH analogues on apoptosis and expression of Bcl-2, Bax, Fas and FasL proteins in endometrial epithelial cell cultures from patients with endometriosis and controls. Human Reproduction 22, 644 653. Chou CS, Tai CJ, MacCalman CD, Leung PC 2003a Dosedependent effects of gonadotropin releasing hormone on matrix metalloproteinase (MMP)-2, and MMP-9 and tissue specific inhibitor of metalloproteinases-1 messenger ribonucleic acid levels in human decidual stromal cells in vitro. Journal of Clinical Endocrinology and Metabolism 88, 680 688. Chou CS, Zhu H, MacCalman CD, Leung PC 2003b Regulatory effects of gonadotropin-releasing hormone (GnRH) I and GnRH II on the levels of matrix metalloproteinase (MMP)-2, MMP-9, and tissue inhibitor of metalloproteinases-1 in primary cultures of human extravillous cytotrophoblasts. Journal of Clinical Endocrinology and Metabolism 88, 4781 4790. Chou CS, Zhu H, Shalev E et al. 2002 The effects of gonadotropinreleasing hormone (GnRH) I and GnRH II on the urokinase-type plasminogen activator/plasminogen activator inhibitor system in human extravillous cytotrophoblasts in vitro. Journal of Clinical Endocrinology and Metabolism 87, 5594 5603. Cunha-Filho JS, Lemos N, Stein N et al. 2005 Vascular endothelial growth factor and inhibin A in follicular fluid of infertile patients who underwent in-vitro fertilization with a gonadotropin-releasing hormone antagonist. Fertility and Sterility 83, 902 907. Eldar-Geva T, Zylber-Haran E, Babayof R et al. 2007 Similar outcome for cryopreserved embryo transfer following GnRH-antagonist/ GnRH-agonist, GnRH-antagonist/HCG or long protocol ovarian stimulation. Reproductive BioMedicine Online 14, 148 154. Griesinger G, Felberbaum R, Diedrich K 2005 GnRH antagonists in ovarian stimulation: a treatment regimen of clinicians second choice? Data from the German national IVF registry. Human Reproduction 20, 2373 2375. Gründker C, Günthert AR, Millar RP, Emons G 2002 Expression of gonadotropin-releasing hormone II (GnRH-II) receptor in human endometrial and ovarian cancer cells and effects of GnRH-II on tumor cell proliferation. Journal of Clinical Endocrinology and Metabolism 87, 1427 1430. Guzeloglu-Kayisli O, Basar M, Arici A 2007 Basic aspects of implantation. Reproductive BioMedicine Online 15, 728 739. Hernandez ER 2000 Embryo implantation and GnRH antagonists: embryo implantation: the rubicon for GnRH antagonists. Human Reproduction 15, 1211 1216. Hughes EG, Fedorkow DM, Daya S et al. 1992 The routine use of gonadotropin-releasing hormone agonists prior to in-vitro fertilization and gamete intrafallopian transfer: a meta-analysis of randomized controlled trials. Fertility and Sterility 58, 888 896. Kol S, Lightman A, Hillensjo T et al. 1999 High doses of gonadotrophin-releasing hormone antagonist in in-vitro fertilization cycles do not adversely affect the outcome of subsequent freeze-thaw cycles. Human Reproduction 14, 2242 2244. Kolibianakis E, Bourgain C, Albano C et al. 2002 Effect of ovarian stimulation with recombinant follicle-stimulating hormone, gonadotropin releasing hormone antagonists, and human chorionic gonadotropin on endometrial maturation on the day of oocyte pickup. Fertility and Sterility 78, 1025 1029. Loumaye E 1990 The control of endogenous secretion of LH by gonadotrophin-releasing hormone agonists during ovarian hyperstimulation for in-vitro fertilization and embryo transfer. Human Reproduction 5, 357 376. Luo X, Ding L, Chegini N 2004 Gonadotropin-releasing hormone and TGF-beta activate MAP kinase and differentially regulate fibronectin expression in endometrial epithelial and stromal cells. American Journal of Physiology Endocrinology and Metabolism 71
287, E991 E1001. Medved R, Virant-Klun I, Meden-Vrtovec H, Tomazevic T 2006 Outcome of frozen-thawed blastocysts derived from gonadotropin releasing hormone agonist or antagonist cycles. Journal of Assisted Reproduction and Genetics 23, 275 279. Meresman GF, Bilotas M, Buquet RA et al. 2003 Gonadotropinreleasing hormone agonist induces apoptosis and reduces cell proliferation in eutopic endometrial cultures from women with endometriosis. Fertility and Sterility 80 (Suppl. 2), 702 707. Raga F, Casan EM, Kruessel J et al. 1999 The role of gonadotropin releasing hormone in murine preimplantation embryonic development. Endocrinology 140, 3705-3712. Ruan HC, Zhu XM, Luo Q et al. 2006 Ovarian stimulation with GnRH agonist, but not GnRH antagonist, partially restores the expression of endometrial integrin beta3 and leukaemiainhibitory factor and improves uterine receptivity in mice. Human Reproduction 1, 2521 2529. Saadat P, Boostanfar R, Slater CC et al. 2004 Accelerated endometrial maturation in the luteal phase of cycles utilizing controlled ovarian hyperstimulation: impact of gonadotropin-releasing hormone agonists versus antagonists. Fertility and Sterility 82, 167 171. Seelig AS, Al-Hasani S, Katalinic A et al. 2002 Comparison of cryopreservation outcome with gonadotropin-releasing hormone agonists or antagonists in the collecting cycle. Fertility and Sterility 77, 472 475. Simon C, Oberyé J, Bellver J et al. 2005 Similar endometrial development in oocyte donors treated with either high- or standard-dose GnRH antagonist compared to treatment with a GnRH agonist or in natural cycles. Human Reproduction 20, 3318 3327. Sirayapiwat P, Suwajanakorn S, Triratanachat S, Niruthisard S 2007 The effects of GnRH antagonist on the endometrium of normally menstruating women. Journal of Assisted Reproduction and Genetics 24, 579 586. Smitz J, Van Den Abbeel E, Bollen N et al. 1992 The effect of gonadotrophin-releasing hormone (GnRH) agonist in the follicular phase on in-vitro fertilization outcome in normo-ovulatory women. Human Reproduction 7, 1098 1102. Ulug U, Turan E, Tosun SB et al. 2007 Comparison of preovulatory follicular concentrations of epidermal growth factor, insulin like growth factor-1, and inhibins A and B in women undergoing assisted conception with gonadotropin releasing hormone agonists and antagonists. Fertility and Sterility 87, 995 998. Vani S, McDonald SE, Williams AR et al. 2007 Mid-luteal endometrial intracrinology following controlled ovarian hyperstimulation involving use of a gonadotrophin releasing hormone antagonist. Human Reproduction 22, 2981 2991. Vlahos NF, Lipari CW, Bankowski B et al. 2006 Effect of lutealphase support on endometrial L-selectin ligand expression after recombinant follicle-stimulating hormone and ganirelix acetate for in-vitro fertilization. Journal of Clinical Endocrinology and Metabolism 91, 4043 4049. Zikopoulos K, Kolibianakis EM, Camus M et al. 2004 Duration of gonadotropin-releasing hormone antagonist administration does not affect the outcome of subsequent frozen-thawed cycles. Fertility and Sterility 81, 473 475. Declaration: The authors report no financial or commercial conflicts of interest. Received 20 March 2008; refereed 14 April 2008; accepted 1 August 2008. 72