ORIGINAL ARTICLE Reproductive endocrinology

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1 Human Reproduction, Vol.24, No.11 pp , 2009 Advanced Access publication on August 11, 2009 doi: /humrep/dep290 ORIGINAL ARTICLE Reproductive endocrinology Does the estradiol level on the day of human chorionic gonadotrophin administration have an impact on pregnancy rates in patients treated with rec-fsh/gnrh antagonist? D. Kyrou 1,5, B. Popovic-Todorovic 1, H.M. Fatemi 1, C. Bourgain 2, P. Haentjens 3, L. Van Landuyt 4, and P. Devroey 1 1 Centre for Reproductive Medicine, Dutch-Speaking Free University Brussels, Laarbeeklaan 101, 1090 Brussels, Belgium 2 Department of Pathology, Dutch-Speaking Free University Brussels, Laarbeeklaan 101, 1090 Brussels, Belgium 3 Centre for Outcomes Research, Dutch- Speaking Free University Brussels, Laarbeeklaan 101, 1090 Brussels, Belgium 4 Department of Embryology, Dutch-Speaking Free University Brussels, Laarbeeklaan 101, 1090 Brussels, Belgium 5 Correspondence address. V.U.B/C.R.G., Laarbeeklaan 101, 1090 Brussels, Belgium. Tel: þ ; Fax: þ ; mimikyrou@yahoo.gr background: The purpose of this prospective observational study was to evaluate the association between estradiol (E 2 ) levels on the day of human chorionic gonadotrophin (hcg) administration and pregnancy rates in a recombinant FSH (rec-fsh) antagonist fixed protocol. methods: A group of 207 patients (39 years of age), treated by IVF/ICSI, received 200 IU/day rec-fsh from Day 2 of the cycle and daily GnRH antagonist starting on Day 6 of stimulation. The criteria for hcg administration included only the presence of 3 follicles of 17 mm diameter. One to two embryos were transferred on Day 3 after oocyte retrieval. results: The area under the curve (AUC) for E 2 on the day of hcg could not distinguish between pregnant and non-pregnant women (AUC:0.5; 95% confidence interval (CI): ). No significant difference was observed between the three percentile groups of E 2 values on the day of hcg administration [group 1, lower 25th percentile (,1142 pg/ml); group 2, medium 50th percentile ( pg/ml) and group 3, higher 75th percentile (.2446 pg/ml)] for the ongoing pregnancy rates (P ¼ 0.52). On the contrary, the linear regression model showed that higher E 2 values on the day of hcg administration significantly improved the scores of transferred embryos (P ¼ 0.01) as well as the total embryo score (P ¼ 0.02). Yet subgroup analysis only in this high responders group revealed lower E 2 and progesterone levels on the day of hcg in pregnant women compared with the non-pregnant (P ¼ 0.01). conclusions: E 2 concentrations on the day of hcg administration in GnRH antagonist cycles are not associated with pregnancy rates. A potential deleterious impact of estradiol on endometrial receptivity is shown for the high responders who have high E 2 levels and improved embryo quality without a concomitant rise in pregnancy rate. Key words: estradiol / GnRH antagonist / IVF / pregnancy rate / implantation Introduction Supraphysiological serum estradiol levels are inextricably related to the ovarian stimulation that is necessary for multiple follicular developments. The E 2 levels can be increased more than 10-fold compared with those found during spontaneous cycles (Pittaway and Wentz, 1983). On the basis of the fact that cyclic changes in the endometrium are regulated by ovarian steroid hormones, the increased E 2 levels due to controlled ovarian hyperstimulation may compromise endometrial receptivity for embryo implantation (Garcia et al., 1984; Hadi et al., 1994). Assessment of the association between estradiol levels on the day of human chorionic gonadotrophin (hcg) administration and pregnancy achievement in in-vitro fertilization (IVF) cycles has been the focus of interest for many years. Adverse effects of supraphysiological serum E 2 can include alteration in both endometrial receptivity and oocyte/embryo quality. However, the detrimental effect of & The Author Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please journals.permissions@oxfordjournals.org

2 Pregnancy rates and E 2 levels on day of hcg 2903 high E 2 levels on uterine receptivity has been subjected to dispute. Simon et al. (1995) has suggested that high E 2 levels are responsible for cases of impaired endometrial receptivity without affecting embryo quality in oocyte donation cycles, although Sharara and McClamrock (1999) have failed to find a detrimental effect on pregnancy outcome of high E 2 levels on the day of hcg administration. To investigate a deleterious effect of E 2 on embryonic implantation, an established in-vitro model for embryonic adhesion was used by Valbuena et al. (2001). Higher E 2 concentration affected embryonic adhesion although a direct toxic effect on the embryo seemed to occur at the cleavage stage. The systematic review by Kosmas et al. (2004) has shown that E 2 levels do not affect treatment outcome in GnRH agonist downregulated IVF/ICSI cycles. However, the role of E 2 levels on the day of hcg administration in pregnancy achievement has only been evaluated retrospectively. The purpose of this prospective observational study was to evaluate the association of E 2 levels on the day of hcg with pregnancy rates and embryo quality in a recombinant FSH (rec-fsh) antagonist fixed protocol. The criteria for hcg administration did not include E 2 levels and were only based on the sonographically confirmed presence of 3 follicles of 17 mm diameter (Kolibianakis et al., 2004). In order to exclude the effects of a prolonged culture period on the embryo quality (Chen et al., 2003) and to investigate the E 2 effect on the outcome, one to two embryos were transferred on Day 3. Materials and Methods Patient population There were 230 patients, treated by IVF/ICSI at the Centre for Reproductive Medicine of the Dutch-Speaking Brussels Free University between October 2007 and December 2008, included in the study. Patients could enter the study only once. Inclusion criteria were age 39 years, body mass index (BMI) between 18 and 29 kg/m 2, presence of both ovaries and basal levels of estradiol (80 pg/ml) and progesterone (1.6 ng/ml) on Day 1 of the cycle. Exclusion criteria were the presence of endometriosis stage 3 (American Fertility Society), polycystic ovarian syndrome (Rotterdam criteria), the need for preimplantation genetic diagnosis and azoospermia testicular sperm extraction. Ovarian stimulation Recombinant FSH (Puregon; NV Organon, Oss, The Netherlands) was started on Day 2 of the menstrual cycle at a dose of 200 IU/day in all patients. The dose of rec-fsh remained unchanged during stimulation until Day 10 of the cycle. If it was necessary to increase the dose of rec-fsh after 10 days of stimulation, or to decrease the dose of rec-fsh due to a risk of Ovarian Hyperstimultion Sydrome (OHSS), the patient was dropped out of the study. To inhibit a premature LH surge, daily GnRH antagonist (Orgalutran; NV Organon) was used from the morning of Day 6 of stimulation. Final oocyte maturation was achieved with IU of hcg (Pregnyl; Organon) as soon as three follicles 17 mm were present on ultrasound (US) scan. Oocyte retrieval was carried out 36 h after hcg administration by transvaginal US-guided puncture of follicles. Conventional IVF or ICSI was carried out as described by Van Landuyt et al. (2005). Oocytes and embryos were cultured in sequential media at 378C in an atmosphere of 6% CO 2,5%O 2 and 89% N 2. The results of the study by Chen et al. (2003) support that increasing E 2 levels on the day of hcg administration are associated with improved pregnancy rates when embryo transfer is performed on Day 5. The increase in pregnancy rates could be potentially favoured by other factors such as a more synchronized supportive uterine environment. One or two embryos were therefore transferred on Day 3 after oocyte retrieval. Embryo selection for transfer or freezing was done in the morning of the day of transfer. Embryos were classified as top quality (score 3), medium quality (score 2) or poor quality (score 1) embryos. Supernumerary top or medium quality embryos were frozen on Day 3. Top quality embryos on Day 3 were defined as embryos with at least eight blastomeres on that morning and with,10% fragmentation. Medium quality embryos were embryos with at least 8 cells and 10 30% fragmentation or 6 7-cell embryos with 0 20% fragmentation. Poor quality embryos were defined as embryos with 6 7 cells and 20 50% fragmentation or 4 5-cell embryos and up to 50% fragmentation. Luteal supplementation The luteal phase was supplemented with vaginal administration of 600 mg natural micronized progesterone in three separate doses (Utrogestan; Piette, Brussels, Belgium) starting 1 day after oocyte retrieval and continuing for 14 days. Hormonal measurements Hormonal assessment was performed at the initiation of stimulation, on Day 6 of rec-fsh stimulation, on Day 8 and on the day of hcg administration. Additional blood samples were taken as necessary between antagonist initiation and hcg administration. Serum LH, FSH, E 2, P and hcg were measured by means of the automated Elecsys immunoanalyser (Roche Diagnostics, Mannheim, Germany). Intra-assay and interasssay coefficients of variation (CVs) were,3 and,4% for LH,,3 and,6% for FSH,,5 and,10% for E 2,,3 and 5% for P and,5 and,7% for hcg, respectively. US assessment US was performed on Day 6 of stimulation and thereafter as necessary in order to ensure that hcg would be injected on the first day that the patient had 3 follicles of 17 mm. At 7 weeks after embryo transfer, a US was performed and the presence of an intrauterine gestational sac confirmed a clinical pregnancy. An US at 12 weeks of gestation defined the ongoing pregnancy. Outcome measures The primary outcome measure was to assess the predictive value of E 2 on the ongoing pregnancy rate and secondary outcome measures were the number of COCs retrieved, embryo quality and the implantation rate (as defined from dividing the total number of fetal hearts observed by US by the number of embryos transferred). Power analysis No data were available in rec-fsh/gnrh antagonist cycles to accurately estimate sample size for the present study. As a consequence, a power analysis could not be performed in this prospective observational study. It was arbitrarily decided that an analysis would be planned as soon as 230 patients had been included.

3 2904 Kyrou et al. Statistical analysis Nominal variables were analyzed in the form of a frequency table by the use of the x 2 test. Normally distributed metric variables were analyzed by the independent sample t-test. Receiver operating characteristic (ROC) curve analysis was performed to assess the predictive value of E 2 day hcg on pregnancy achievement. Tukey s Hinges percentile analysis used to define groups of E 2 on hcg day. One-way analysis of variance (ANOVA) with least significant difference post hoc test was used to compare the three percentile groups. Multiple linear and logistic regressions were used to identify the variables that may contribute to the outcomes whereas adjusting for confounding variables (the parameters could enter the model only if they were significantly different between pregnant and non-pregnant patients). All tests were two-tailed, with a confidence level of 95% (P, 0.05). Values are expressed as mean (SD), unless stated otherwise. Results There were 230 patients started stimulation. For seven patients, due to a lack of response, an increase in the dose of rec-fsh was necessary and therefore they were excluded from the study. Additionally, 16 patients dropped out because a decrease in the dose of rec-fsh was necessary due to risk of OHSS. Finally, 207 patients were further analysed. The women included in this study were aged years. The serum E 2 concentrations on the day of hcg administration were on average pg/ml and the mean number of oocytes obtained was The ongoing pregnancy rate was 29.5% (61/207): 55 were singleton and six were twin pregnancies. Table I summarizes the patient characteristics, stimulation and treatment outcome parameters of the pregnant compared with nonpregnant women. There was no significant difference in E 2 levels on the day of hcg between pregnant and non-pregnant women (P ¼ 0.40). Pregnant patients had significantly lower FSH and LH levels on the day of hcg administration compared with the non-pregnant patients (13.8 versus 15.3, P ¼ 0.01; 1.5 versus 2.4, P ¼ 0.01). Logistic regression using as the dependent variable the achievement of ongoing pregnancy and as independent variables the basal FSH, and LH, progesterone and FSH on the day of hcg is shown in Table II. A significant effect on the achievement of ongoing pregnancy was observed for the basal FSH (0.84, 95% confidence interval (CI): ) and the progesterone level on day hcg (0.52, 95% CI: ). The lower the level of FSH at the initiation of the stimulation and the lower the level of progesterone on the day of hcg administration, the higher the probability of achievement of ongoing pregnancy. The lack of an effect of the E 2 levels on the hcg day on the pregnancy rate was confirmed with logistic regression. To assess the predictive value of serum E 2 level on the day of hcg administration on pregnancy achievement, a ROC curve analysis was performed (Fig. 1). The area under the curve (AUC) calculated could not distinguish between pregnant and non-pregnant women (AUC:0.50; 95% CI: ). Percentile analysis to define percentile groups of E 2 on the day hcg was performed. Table III show patients characteristics and outcomes according to three groups of estradiol values on the day of hcg as follows: group 1, lower 25th percentile (,1142 pg/ml); group 2, medium 50th percentile ( pg/ml) and group 3, higher Table I Differences in patient s characteristics and stimulation data among pregnant and not pregnant women Pregnant Not pregnant P (n 5 61) (n 5 146)... Age (years) 32.4 (3.7) 33.2 (3.7) 0.20 BMI (kg/m 2 ) 23.4 (3.6) 23.1 (3.1) 0.66 No. of trials 1.1 (0.2) 1.2 (0.6) 0.77 Cause of infertility (%) 0.34 Male Ovulatory Tubal Idiopathic Lesbian Treatment (%) 0.84 IVF ICSI Stimulation Duration of stimulation 9.1 (1.5) 9.3 (1.6) 0.38 (days) Total FSH administered (301) (315) 0.38 (IU) Duration of antagonist 5.2 (1.6) 5.3 (1.5) 0.61 treatment (days) Basal FSH (IU/l) 7 (2.3) 8.2 (2.7) 0.01 Basal E 2 (pg/ml) 38 (15.1) 40.5 (17.3) 0.34 E 2 on Day 6 (pg/ml) (325) (392.7) 0.94 E 2 on Day 8 (pg/ml) (703.6) (729.8) 0.81 E 2 on hcg day (pg/ml) (947.2) (1334.9) 0.40 LH on day of hcg (IU/l) 1.5 (1.5) 2.4 (2.9) 0.01 Progesterone on day of 1.1 (0.6) 1.2 (0.6) 0.09 hcg (ng/ml) FSH on hcg day (IU/l) 13.8 (4.1) 15.3 (3.9) 0.01 Treatment outcome No. COCs retrieved 12.2 (6.3) 11.5 (7.2) 0.54 No. embryos transferred 1.6(0.5) 1.5 (0.5) 0.56 Total embryo score 9.5 (10.5) 8.5 (8.1) 0.48 Values are means (standard deviation). BMI ¼ body mass index; ICSI ¼ intracytoplasmic sperm injection; FSH ¼ follicle-stimulating hormone; LH ¼ luteinizing hormone; E 2 ¼ estradiol; hcg ¼ human chorionic gondotrophin; COCs ¼ cumulus- oocyte complexes. 75th percentile (.2446 pg/ml). There were no statistically significant differences between the three groups in the implantation rates, ongoing pregnancy rates and the number of pregnancies. A post-hoc analysis revealed that significant difference existed between medium ( pg/ml) and higher (.2446 pg/ml) percentile in the duration of the stimulation, the amount of FSH administered, the duration of antagonists treatment and the serum progesterone on the day of hcg. The basal E 2 was different only between the lower (,1142 pg/ml) and medium ( pg/ml) percentile. Although the difference in the scores of the embryos transferred

4 Pregnancy rates and E 2 levels on day of hcg 2905 Table II Logistic regression on achievement of ongoing pregnancy after embryo transfer Dependent variable: P-value Odds 95% CI... achievement of ongoing ratio Lower Upper pregnancy... Independent variables Basal FSH LH on hcg day P on hcg day FSH on hcg day Model P ¼ 0.01 Hosmer and Lemeshow Test P ¼ 0.93 CI ¼ confidence interval. Figure 1 ROC curve for AUC E 2 on hcg day with dependent parameter the achievement of ongoing pregnancy. was significant between the lower (,1142 pg/ml) and the medium ( pg/ml) percentile and between the lower (,1142 pg/ml) and the higher (.2446 pg/ml) percentile, the difference in the total scores of the embryos obtained was significant only between lower (,1142 pg/ml) and higher (.2446 pg/ml) percentiles. A subgroup analysis was performed, comparing E 2 on the day of hcg in the pregnant and not pregnant women in only the high responders group (Table IV). E 2 levels on the day of hcg were significantly lower in pregnant women compared with the non-pregnant (P ¼ 0.01). Pregnant patients also had significantly lower progesterone and LH levels on the day of hcg administration compared with the non-pregnant patients (1.1 versus 1.5, P ¼ 0.01; 1.2 versus 2.1, P ¼ 0.02). To assess the predictive value of E 2 on the day of hcg and other independent variables (treatment, indications, age) on the total score of the embryos, the scores of the embryos transferred and cryopreserved, a linear regression model was applied. Serum E 2 on day of hcg was found to be positively correlated with embryo quality of the total embryos (r 2 ¼ 0.8, P ¼ 0.001), of the embryos transferred (r 2 ¼ 0.02, P ¼ 0.02) and of the cryopreserved embryos (r 2 ¼ 0.08, P ¼ 0.001). Among the other parameters, age had a negative correlation only with the embryo quality of the transferred embryos (r 2 ¼ 0.02, P ¼ 0.001). Discussion To the best of our knowledge, this is the first study to prospectively investigate the association of E 2 levels on the day of hcg and pregnancy achievement in GnRH antagonist down-regulated IVF/ICSI treatment cycles, since previously reported studies had been conducted retrospectively in GnRH agonist cycles. According to our findings, low basal FSH and low progesterone level on the day of hcg are independent predictors of pregnancy achievement. In addition, our results have shown that the concentration of estradiol on the day of hcg administration does not interfere with the occurrence of ongoing pregnancies. Regarding the FSH levels, our results based on the logistic regression analysis are in agreement with the only study of GnRH antagonist cycles by Jurema et al. (2003), which has shown that lower basal levels of FSH are correlated with improved ovarian response and pregnancy rates in IVF cycles. Basal FSH levels are one of the most commonly used tests for predicting success in IVF treatment as first described by Muasher et al. (1988). Numerous studies have shown that an elevated basal FSH levels are associated with lower pregnancy rates in ART (Scott et al., 1989; Martin et al., 1996; Sharif et al., 1998). Additionally, a systematic review of tests predicting IVF outcome by Broekmans et al. (2006) has shown that the measurement basal FSH in regularly cycling women is accurate in the prediction of non-pregnancy only at very high threshold levels. The authors recommend its use as a screening test for counselling purposes. Our data show that a rise in progesterone on the day of hcg administration impairs pregnancy outcome. Premature luteinization refers to a rise in serum progesterone levels on the day of hcg administration above a threshold level, and its incidence is between 5 and 30% in IVF patients (Schoolcraft et al., 1991; Silverberg et al., 1991; Fanchin et al., 1993; Ubaldi et al., 1995). A recent systematic review and meta analysis by Venetis et al. (2007) on this issue analysed mostly GnRH agonist cycles (GnRH antagonists: n ¼ 109, GnRH agonists: n ¼ 2624). The results suggest that progesterone elevation, on the day of hcg administration for final oocyte maturation, does not appear to be associated with the probability of pregnancy in women undergoing ovarian stimulation with GnRH analogues and gonadotrophins for IVF. A more recent publication by our group (Papanikolaou et al., 2009) after using a GnRH antagonist/rec-fsh protocol has shown that even modest rises of progesterone in the follicular phase have detrimental effect on the implantation potential of a good-quality cleavage stage embryo. In the case of estradiol, our results showed no association between E 2 levels and pregnancy achievement, which is in agreement with the meta-analysis by Kosmas et al. (2004). The studies included in this meta-analysis differ in their conclusions: two studies (Chenette et al., 1990; Gelety and Buyalos, 1995) show a positive association, two studies show a negative association of E 2 and pregnancy rates (Simon et al., 1995; Ng, 2000), and the majority of studies show no association (Dor et al., 1992; Sharara and McClamrock, 1999; Papageorgiou et al., 2002; Chen et al., 2003). The controversy of this issue might be partially explained by the retrospective design of

5 2906 Kyrou et al. Table III Percentile groups of E 2 on the day of hcg administration and their effects on different parameters and outcomes Lower 25th percentile Medium 50th percentile Higher 75th percentile P (<1142 pg/ml) (n 5 51) ( pg/ml) (n 5 105) (>2446 pg/ml) (n 5 51)... Age (years) 32.6(3.7) 33.4 (3.5) (4.1) 0.21 BMI (kg/m 2 ) 23.6 (3.7) 23 (3.2) 23.5 (3.3) 0.49 No. of trials 1.2 (0.539) 1.1 (0.6) 1.1 (0.38) 0.72 Cause of infertility (%) 0.16 Male Ovulatory Tubal Idiopathic Lesbian Treatment (%) 0.61 IVF ICSI Duration of stimulation (days) 9.3 (1.7) 9.1 (1.5) 9.8 (1.4) 0.02** Total FSH administered (IU) (346) (293.4) (291) 0.02** Duration of antagonist treatment (days) 5.3 (1.7) 5.1 (1.5) 5.7 (1.4) 0.04** Basal FSH (IU/l) 8.5 (3.6) 7.9 (2.3) 6.9 (2) 0.07 Basal E 2 (pg/ml) 34.2 (16.5) 42.2 (18.4) 40.4 (11.8) 0.02* Basal LH (IU/L) 5 (2.9) 5.2 (2.1) 6.1 (2.2) 0.04 Progesterone on hcg day (ng/ml) 1.2 (0.8) 1.2 (0.6) 1.4 (0.5) 0.04** Endometrium on day hcg 9 (1.5) 9.5 (2.1) 9.6 (2) 0.30 No. COCs retrieved 7.7 (4.8) 11.1 (5.5) 17.2 (8) 0.01**** No of embryos transferred 1.4 (0.5) 1.5 (0.5) 1.6 (0.5) 0.06 Score of embryos transferred 3.2 (1.5) 3.9 (1.6) 4.1 (1.5) 0.01*/*** Score of embryos frozen 3 (4.4) 4.8 (7.1) 7.9 (12.3) 0.05 Total embryo score 5.8 (4.9) 8.7 (7.6) 12 (12.6) 0.02*** Implantation rate (%) Ongoing pregnancy rate (%) No of pregnancies (%) 0.64 Singletons Twins Data are presented as mean (SD). P* value correspond on post-hoc analysis between lower and medium percentile. P** value correspond on post-hoc analysis between medium and upper percentile. P*** value correspond on post-hoc analysis between lower and upper percentile. P**** correspond on post-hoc analysis between the three percentiles. the studies, small sample sizes, as well as methodological issues such as the inclusion of more than one cycle for analysis of the same patients in more than three studies (Kosmas et al., 2004). E 2 levels are routinely assessed during ovarian stimulation by a number of clinics, even though they are not used as a criterion for hcg administration. The decision on timing for inducing final oocyte maturation is mostly based on sonographic evaluation of the number and size of the follicles. The use of E 2 levels to distinguish poor, normal and high responders in order to predict treatment outcome is still controversial (Papageorgiou et al., 2002). Different E 2 cut-off levels (Simon et al., 1995, 1998; Ng, 2000) make interpretation of data cumbersome. Despite the fact that percentile curves present a more objective tool, there are still no uniformly accepted E 2 percentiles. Papageorgiou et al., (2002) have used 10th and 90th percentiles to define the type of response, with normal responders having E2 between the 10th and 90th percentile. In the present study, we have used percentile analysis to divide the patients in three groups according to E 2 levels on day of HCG administration (,1142, ,.2446 pg/ml). Our results show that in the higher 75th percentile (E 2 levels.2446 pg/ml), the pregnancy rates remained the same as compared with the medium and lower percentile group, although the embryo score was higher than the two other groups. These patients had significantly higher progesterone levels on the day of hcg, significantly more oocytes were retrieved than from the other two groups and their embryos, both transferred and frozen, had higher scores. The association between the high E 2 and premature progesterone elevation suggest that at least one of the mechanisms that play a role in the premature increase of plasma progesterone is

6 Pregnancy rates and E 2 levels on day of hcg 2907 Table IV Comparison between different parameters for pregnant and non-pregnant women in high responders group (E 2 > 2446 pg/ml) Pregnant (n 5 15) Not pregnant (n 5 36) P... Age (years) 32.5 (4.5) 32.3 (3.9) 0.90 BMI (kg/m 2 ) 23.3 (3.3) 23.5 (3.3) 0.90 No. of trials 1 (0.2) 1.1 (0.4) 0.54 Cause of infertility (%) 0.15 Male Ovulatory Tubal Idiopathic Lesbian 0 0 Treatment (%) 0.06 IVF ,1 ICSI Stimulation Duration of stimulation (days) 10 (1.7) 9.6 (1.3) 0.46 Total FSH administered (IU) 2000 (354) 1933 (262) 0.46 Duration of antagonist treatment (days) 6 (1.7) 5.5 (1.2) 0.34 Basal FSH (IU/l) 6.1 (1.6) 7.2 (2.1) 0.07 Basal E 2 (pg/ml) 42.6 (15) 39.4 (10.2) 0.38 E 2 on Day 6 (pg/ml) (326.8) (420.5) 0.20 E 2 on Day 8 (pg/ml) (887) 1941 (833) 0.55 E 2 on hcg day (pg/ml) (636) 3933 (1209) 0.01 LH on day of hcg (IU/L) 1.2 (0.7) 2.1 (1.9) 0.02 Progesterone on day of hcg (ng/ml) 1.1 (0.4) 1.5 (0.4) 0.01 FSH on hcg day (IU/l) 12.9 (3.5) 13.7 (3.9) 0.48 Treatment outcome No. COCs retrieved 16.6 (6.8) 17.5 (8.5) 0.71 No. embryos transferred 1.6 (0.5) 1.7 (0.4) 0.79 Quality score of transferred embryos 4.2 (1.6) 4 (1.4) 0.7 Values are means (standard deviation). linked to the high response of the ovary to ovarian stimulation. An excess in the number of follicles and consecutively an excess of proliferating granulosa cells can lead to an increased progesterone production. The fact that the embryos were of better quality in the highresponder group, with progesterone elevation, makes the theory of impaired oocyte/embryo quality unlikely. Instead, the high follicular progesterone, can advance into the endometrium and therefore the replacement of a Day 3 embryo (earlier than when it happens naturally) in an asynchronous endometrium may result in implantation failure (Papanikolaou et al., 2009). In combination with the premature elevation of progesterone, the high E2 levels had an negative effect, since transfer of high quality embryos in cycles with high E 2 on day of hcg did not result in pregnancy achievement. Chen et al. (2007) have evaluated 1196 GnRH agonist cycles grouped by peak E 2 percentile distribution into three groups: normal responder between percentile 25 75: pg/ml, n ¼ 595 cycles; moderate high responders P75 P90, E 2 between pg/ml, n ¼ 180 cycles; and for the high response group, the E 2 cut-off concentration was set as P90 and above E pg/ml, n ¼ 119 cycles. The high response group showed decreased trends in implantation and pregnancy rates compared with normal responders, but statistical significance was reached only for the difference in implantation rates. High serum estrogen levels were detrimental to implantation, but not to the quality of oocytes, which may be due to an adverse effect on endometrial receptivity in ovarian stimulation cycles. On the basis of our results and the findings by Chen et al. (2007), in order to improve pregnancy rates in the case of high estradiol levels at the end of the ovarian stimulation, a proposed strategy could be the selection for embryo transfer on Day 5. Transfer of blastocysts may offer the endometrium time to recover from any negative effects caused by the exposure to high peak E 2 levels. Ovarian stimulation induces supraphysiological levels of steroids during the follicular phase resulting in advanced endometrial

7 2908 Kyrou et al. development regardless of the type of GnRH analogue used. In GnRH-agonist cycles, endometrial biopsies taken in the pre-ovulatory phase prior to hcg injection have shown accentuated proliferative aspects and early secretory changes even before the rise in progesterone occurs (Marchini et al., 1991). Biopsies taken on the day of oocyte retrieval show endometrial advancement in more than 90% of the cases, with no pregnancy occurring if the advancement is exceeded by 3 days (Ubaldi et al., 1997). These findings have been confirmed in GnRH-antagonist cycles (Kolibianakis et al., 2002). A study by Van Vaerenbergh et al. (2009) on endometrial gene expression on the day of oocyte retrieval in antagonist cycles with embryo replacement on Day 3 also confirmed the histologically advanced endometrial maturation. A lower endometrial receptivity was observed in patients with histological dating exceeding 3 days advancement. Increased sensitivity to progesterone resulting in secretory advancement could be induced by elevated estrogen concentration (Jacobs et al., 1987). In GnRH-agonist stimulated cycles, moderate responders exhibit less desynchrony in endometrial glandular and stromal maturation compared with high responders (Basir et al., 2001). However, very little data are available on the role of E 2 on the endometrial receptivity in GnRH-antagonist stimulated cycles. Detti et al. (2008a) found no correlation in endometrial US dynamics, E 2 concentrations and pregnancy outcome. Decreased endometrial thickness in these cycles was related to miscarriage rate but not to estradiol concentrations (Detti et al., 2008b). In our study the absolute number of oocytes obtained was proportional to the E 2 on hcg day, with reduced numbers of COCs from the higher to the lower percentiles for E 2 levels. However, as the number of oocytes retrieved increased, the implantation and pregnancy rates remained unchanged, no decreases were observed, in contradiction with previous studies (Forman et al., 1988; Pellicer et al., 1989). An explanation for the lack of effect on implantation and pregnancy rates could be the low number of very high responder women in the study. In conclusion, although improved embryo quality has been observed in the presence of high E 2 levels, no subsequent increase in pregnancy rate was noticed. This observation suggests that elevated E 2 concentrations could have a deleterious effect on endometrial receptivity. An increase in progesterone during the follicular phase, which is also a reflection of a good ovarian response, can also have a negative impact on the endometrium. The results of this study render further molecular and clinical research to establish the degree of endometrial advancement with the regard to the follicular estradiol levels in the GnRH antagonist cycles. References Basir GS, WS O, Ng EH, Ho PC. Morphometric analysis of peri-implantation endometrium in patients having excessively high oestradiol concentrations after ovarian stimulation. Hum Reprod 2001; 16: Broekmans FJ, Kwee J, Hendriks DJ, Mol BW, Lambalk CB. A systematic review of tests predicting ovarian reserve and IVF outcome. Hum Reprod Update 2006;12: Chen CH, Zhang X, Barnes R, Confino E, Milad M, Puscheck E, Kazer RR. Relationship between peak serum E2 levels and treatment outcome in in-vitro fertilization cycles after embryo transfer on day 3 or day 5. Fertil Steril 2003;80: Chen QJ, Sun XX, Li L, Gao XH, Wu Y, Gemzell-Danielsson K, Cheng LN. 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