Menstruation-free interval and ongoing pregnancy in IVF using GnRH antagonists

Human Reproduction Vol.21, No.4 pp. 1012 1017, 2006 Advance Access publication December 8, 2005. doi:10.1093/humrep/dei415 Menstruation-free interval and ongoing pregnancy in IVF using GnRH antagonists Efstratios M.Kolibianakis 1,3, Evangelos G.Papanikolaou 2, Michel Camus 2, Herman Tournaye 2, André C.Van Steirteghem 2 and Paul Devroey 2 1 Unit for Human Reproduction, Aristotle University, Thessaloniki, Greece and 2 Centre for Reproductive Medicine, Vrije Universiteit Brussel, Brussels, Belgium 3 To whom correspondence and reprint requests should be addressed. E-mail: Stratis.Kolibianakis@otenet.gr BACKGROUND: The purpose of this study was to evaluate prospectively the association between the achievement of ongoing pregnancy and the time interval from the end of menstruation until the administration of HCG (menstruationfree interval) in patients treated by IVF. METHODS: A fixed dose of 200 IU of recombinant FSH (rfsh) was started in 90 patients on day 2 of the menstrual cycle and daily GnRH antagonist was initiated on day 6 of stimulation. Triggering of final oocyte maturation was performed with 10000 IU of HCG as soon as three follicles of 17 mm were present at ultrasound. RESULTS: Single embryo transfer was performed in 64.6% of the patients who reached embryo transfer (53/82). Ongoing pregnancy rate per embryo transfer was 18.3% (95% CI 11.4 28.0%). The menstruation-free interval significantly predicted the probability of ongoing pregnancy in a logistic regression analysis, controlling for female age and LH on day 1 of stimulation (odds ratio for the menstruation-free interval: 0.70; 95% CI: 0.54 0.92). CONCLUSION: The longer the interval from the end of menstruation until the administration of HCG, the lower the probability of ongoing pregnancy in patients stimulated with recombinant FSH and GnRH antagonist for IVF. Key words: GnRH antagonists/menstruation-free interval/recombinant FSH/ongoing pregnancy Introduction During the follicular phase of a natural cycle, endometrium is subjected to the action of steroid hormones, which are secreted by the dominant follicle. Following ovulation, an adequately prepared endometrium is exposed to elevated progesterone levels, secreted by the corpus luteum, and acquires properties that will allow implantation. On the contrary, endometrium during the follicular phase of an IVF cycle is exposed to much higher steroid levels, which are secreted by the multiple developing follicles. Thus, during this time period, endometrium accepts a quantitatively different stimulus compared with the natural cycle. This might lead to an abnormal endometrial quality by the end of the follicular phase. Support for this concept exists at the steroid receptor level in GnRH antagonist IVF cycles. It has been recently shown that ovarian stimulation for IVF induces steroid receptor changes, before HCG administration, similar to those observed in the early luteal phase (Papanikolaou et al., 2005). Moreover, before HCG administration, premature secretory endometrial changes have been detected in GnRH agonist IVF cycles (Marchini et al., 1991). On the day of oocyte retrieval, there is also plenty of evidence showing that abnormalities in the endometrium are usually the rule. In most studies performed, endometrial biopsies reveal advancement of endometrium (for review: Kolibianakis and Devroey, 2002). The importance of the changes observed by the end of the follicular phase for the achievement of ongoing pregnancy has recently been shown. In IVF cycles where biopsies were taken on the day of oocyte retrieval and an embryo transfer was performed a few days later, extreme endometrium advancement was associated with a decreased probability of ongoing pregnancy (Kolibianakis et al., 2002). It was subsequently assumed that by prolonging the follicular phase the abnormalities induced by the abnormal steroid levels might be intensified, thereby compromising endometrial receptivity. Indeed, it was shown that prolongation of the follicular phase by 2 days in GnRH antagonist cycles results in a decreased probability of pregnancy (Kolibianakis et al., 2004a). This was not attributed to differences in embryo quality, as assessed by morphological criteria, but to differences in endometrial histology (Kolibianakis et al., 2005). A similar observation has been reported in GnRH agonist cycles where prolongation of the follicular phase was associated with a decreased probability of pregnancy (Clark et al., 1991). If prolonged exposure of endometrium to unnatural levels of steroid hormones results in histological abnormalities and in 1012 The Author 2005. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Menstruation-free interval and ongoing pregnancy in IVF turn decreases pregnancy rates, then duration of stimulation might be negatively associated with pregnancy achievement. However, such an association has so far not been established in the literature. What might have been overlooked, though, is that theoretically in order for hormonal exposure to affect it, endometrium should be ready to accept the hormonal stimulus and progress histologically. Therefore, menstruation should have stopped. From that point onwards, the duration of the interval until the administration of HCG might be more important, as circulating steroids are able to act during this period on the re-epithelialized endometrium. The hypothesis tested in the current prospective study was that the interval from the end of menstruation until the day of HCG administration is associated with the probability of ongoing pregnancy in patients treated for IVF with GnRH antagonists and rfsh. Materials and methods Patient population Ninety patients treated by IVF at the Centre for Reproductive Medicine of the Dutch-speaking Brussels Free University from October 2004 until March 2005 were included in the study. Inclusion criteria were age <39 years, body mass index (BMI) between 18 and 29 kg/m 2, presence of both ovaries, absence of endometriomas detected at ultrasound, FSH levels <10 IU/l and basal levels of estradiol (E 2 <80 pg/ml) and progesterone (<1.6 ng/ml) at initiation of stimulation. Patients could enter the study only once and in order to be analysed were required to have reached embryo transfer. An informed consent was obtained by all patients participating in the study. Ovarian stimulation and IVF procedure Stimulation was performed with recombinant FSH (rfsh, Puregon; NV Organon, Oss, The Netherlands) starting in the afternoon of day 2 of the cycle at 200 IU per day. The dose of rfsh remained unchanged during stimulation. Daily GnRH antagonist 0.25 mg (Orgalutran; NV Organon) was used to inhibit premature LH surge and was always started on the morning of day 6 of stimulation. Final oocyte maturation was achieved by the administration of 10000 IU of HCG (Pregnyl; Organon) as soon as 3 follicles of 17 mm were present on ultrasound. Steroid levels were measured but were not taken into consideration for the administration of HCG, which was based exclusively on follicular development. Oocyte retrieval was carried out 36 h after HCG administration by transvaginal ultrasound-guided puncture of follicles. Conventional IVF was performed in 25 couples, ICSI in 52 couples and both conventional IVF and ICSI in five couples. ICSI and IVF procedures have been described in detail previously (Van Steirteghem et al., 1993; Devroey and Van Steirteghem, 2004; Devroey et al., 1995; Van Landuyt et al., 2005). As a matter of principle, one or two embryos were transferred on day 3 or day 5 after oocyte retrieval. Embryos were classified as top quality (score 1), medium quality (score 2), and low quality (score 3), as previously described (Staessen et al., 1992; Gardner and Schoolcraft, 1999; Van Landuyt et al., 2005). The mean score of the embryos transferred to each patient was used for the calculation of the mean embryo quality score of all embryos transferred. Luteal supplementation The luteal phase was supplemented with the vaginal administration of 600 mg natural micronized progesterone in three separate doses (Utrogestan; Besins, Brussels, Belgium), starting 1 day after oocyte retrieval and continued until 7 weeks of gestation if pregnancy was achieved. Hormonal measurements Hormonal assessment was performed at initiation of stimulation, on day 6, on day 8 of rfsh stimulation and on the day of HCG administration. Additional blood samples were taken as necessary between antagonist initiation and HCG administration. Serum LH, FSH, HCG, E 2 and progesterone levels were measured by means of the automated Elecsys immunoanalyser (Roche Diagnostics, Mannheim, Germany). Intra- and inter-assay coefficients of variation (CV) were <3 and <4% for LH, <3 and <6% for FSH, <5 and <7% for HCG, <5 and <10% for E 2 and <3 and <5% for progesterone respectively. The inter-assay CV for LH at 0.4 IU/l was 6.6% and at 1.0 IU/l was 2.7%. Ultrasound assessment Ultrasound was performed on day 6 of stimulation and thereafter as necessary in order to ensure that HCG was injected on the first day the patient had 3 follicles of 17 mm. For that purpose, a follicular growth of 2 mm per day was assumed to be present (Kolibianakis et al., 2004a). Outcome measures Pregnancies progressing beyond the 12th week of gestation were considered to be ongoing. Ongoing implantation rate was calculated by dividing the number of gestational sacs with fetal heart beat present at 12 weeks of gestation by the number of embryos transferred. The patients were asked to report the first day they were completely free of menstruation. This day was confirmed as such, during follow-up of ovarian stimulation. The menstruation-free interval was calculated from that day until the day of HCG. The 1st day of period was reported by the patient as the first day during which bleeding was present at the time of awakening (pre-menstrual spotting was not considered as indicating initiation of menstruation). Statistical analysis Power analysis No data were available in GnRH antagonist cycles to accurately estimate sample size for the present study and thus a power analysis in this prospective pilot study could not be performed. A first interim analysis was planned arbitrarily after the first 90 patients had been included. Statistical tests Nominal variables were examined with the use of the exact χ 2 for trend. Metric variables were analysed by the Mann Whitney U-test. The effect of menstruation-free interval on ongoing pregnancy achievement after embryo transfer was examined by robust logistic regression. Variables which entered the model were those that were selected by means of univariate comparisons between patients that did or did not achieve an ongoing pregnancy if P < 0.1. All tests were two-tailed with a confidence level of 95% (P < 0.05). Values are expressed as mean ± SEM. Results Patient and stimulation characteristics Ninety patients started stimulation. Cycles were cancelled in three patients due to lack of response to stimulation after 10 days. In addition, two patients were excluded from analysis 1013

E.M.Kolibianakis et al. due to antagonist initiation later than day 6 of stimulation. In a further three patients no embro transfer was performed due to ovarian hyperstimulation syndrome (OHSS) risk. Therefore 82 patients completed an IVF cycle and were further analysed. The mean (± SEM) age of the patients who reached embryo transfer was 32.2 ± 0.4 years. Patients had performed a mean of 1.1 ± 0.2 previous IVF/ICSI trials, while the mean FSH at initiation of stimulation was 7.3 ± 0.2 IU/l. The majority of the couples were treated for male factor infertility (64.6%, n = 53), while 18.3% were treated for tubal infertility (n = 15). In 17.1% of couples (n = 14) no reason for infertility could be identified (unexplained infertility). The mean duration of FSH stimulation was 9.3 ± 0.2 days and the mean total units of rfsh used were 1858 ± 38 IU. A mean of 13.1 ± 0.9 cumulus oocyte complexes (COC) were retrieved and following fertilization a mean of 7.4 ± 0.5 two pronuclear (2PN) oocytes were available (fertilization rate 56.9 ± 2.2%). Single embryo transfer was performed in 64.6% of patients (n = 53). A mean number of 1.4 ± 0.1 embryos were transferred per patient, resulting in a 15.0 ± 3.7% ongoing implantation rate. Ongoing pregnancy rate per oocyte retrieval was 18.3% (95% CI: 11.4 28.0%). Association between the menstruation-free interval and achievement of ongoing pregnancy after oocyte retrieval Figure 1 shows histograms for duration of menstruation, stimulation and menstruation-free interval in patients treated with Table I. Duration (days) of menstruation, stimulation and the menstruation-free interval in IVF patients stimulated with rfsh and GnRH antagonists who did or did not achieve an ongoing pregnancy Ongoing pregnancy (n = 15) Not pregnant (n = 67) Menstruation 5.1 ± 0.2 4.8 ± 0.1 0.249 Stimulation 9.7 ± 0.3 10.4 ± 0.2 0.093 Menstruation-free interval 4.6 ± 0.4 5.6 ± 0.2 0.055 GnRH antagonist and rfsh. The mean duration of menstruation was 4.8 ± 0.1 days, the mean duration of stimulation was 10.2 ± 0.2 days and the mean duration of the menstruation-free interval was 5.4 ± 0.2 days. Table I shows the duration of menstruation, stimulation and menstruation-free interval in patients who did or did not achieve an ongoing pregnancy. Table II shows the comparison between patients who did or did not achieve an ongoing pregnancy for several continuous and nominal variables. Based on these comparisons the menstruation-free interval, the LH at initiation of stimulation and the female age entered the logistic regression model. The duration of stimulation also met the criteria for entering the logistic regression analysis (P < 0.1). However, due to its significant correlation with the menstruation-free interval from which it is derived (Spearman correlation coefficient: 0.86, P < 0.001), the two parameters could not enter the model simultaneously. For this reason, the model was P Figure 1. Histogram for duration of menstruation, stimulation and the menstruation-free interval in IVF patients stimulated with rfsh and GnRH antagonist. 1014

Menstruation-free interval and ongoing pregnancy in IVF Table II. Baseline, stimulation characteristics and embryological data in patients stimulated with rfsh and GnRH antagonists for IVF who did or did not achieve an ongoing pregnancy. Ongoing pregnancy Not pregnant Baseline parameters Female age (years) 30.7 ± 0.5 32.6 ± 0.8 0.064 FSH on day 1 of stimulation (IU/l) 6.9 ± 0.4 7.4 ± 0.2 0.212 LH on day 1 of stimulation (IU/l) 4.7 ± 0.6 5.6 ± 0.2 0.097 No. of previous trials 1.2 ± 0.5 1.1 ± 0.3 0.390 Body mass index (kg/m 2 ) 23.5 ± 0.4 23.1 ± 0.4 0.774 Indication for treatment, n (%) 0.926 Andrological 10 (66.7) 43 (64.2) Tubal 3 (20.0) 12 (17.9) Idiopathic 2 (13.3) 12 (17.9) Hormones on the day of HCG FSH (IU/l) 14.1 ± 1.1 14.6 ± 0.4 0.615 LH (IU/l) 1.6 ± 0.3 1.7 ± 0.2 0.949 Estradiol (pg/ml) 1909 ± 244 2256 ± 150 0.382 Progesterone (ng/ml) 1.3 ± 0.2 1.4 ± 0.1 0.417 Endometrial thickness on the 9.4 ± 0.6 9.7 ± 0.4 0.659 day of HCG (mm) Stimulation outcome Cumulus oocyte complexes retrieved 14.1 ± 2.6 13.2 ± 0.9 0.931 Fertilization rate (%) 63.4 ± 5.1 57.2 ± 2.3 0.279 Two-pronuclear oocytes 8.5 ± 1.6 7.2 ± 0.6 0.471 Embryos transferred 1.4 ± 0.2 1.4 ± 0.1 0.937 Mean embryo quality score 1.3 ± 0.1 1.5 ± 0.1 0.798 Embryos cryopreserved 4.2 ± 1.4 3.1 ± 0.4 0.798 run twice by including each of the two parameters (menstruation-free interval and duration of stimulation) separately, together with female age and LH level on day 1 of stimulation. Robust logistic regression using as dependent variable the achievement of ongoing pregnancy after embryo transfer and as independent variables the female age, the level of LH at initiation of stimulation and the duration of the menstruation-free interval is shown in Table III. A significant effect on the achievement of ongoing pregnancy after embryo transfer was observed for the female age (0.87, 95% CI: 0.76 0.99) and the duration of the menstruation-free interval (odds ratio: 0.70, 95% CI: 0.54 0.92). The higher the patient s age and the longer the duration of the menstruation-free interval, the lower the probability of achieving an ongoing pregnancy after embryo transfer. When the menstruation-free interval was replaced by duration of stimulation, the model just reached significance (P = 0.049); however, neither the female age (P = 0.057) nor the duration of stimulation (P = 0.066) significantly predicted the probability of ongoing pregnancy in this case. Table III. Robust logistic regression on achievement of ongoing pregnancy after embryo transfer in patients stimulated with rfsh and GnRH antagonists Dependent variable: achievement of ongoing pregnancy P Odds ratio 95% CI Lower P Upper Menstruation-free interval 0.010 0.70 0.54 0.92 Age at initiation of stimulation 0.040 0.87 0.76 0.99 LH level on day 1 0.149 0.74 0.49 1.11 Ongoing pregnancy rates per embryo transfer in groups of patients defined according to thirdile analysis of the duration of their menstruation, the duration of their stimulation and the duration of their menstruation-free interval is shown in Table IV. A significant trend toward decreased ongoing pregnancy rates was observed with an increased duration of the menstruationfree interval. Such an association was not observed for duration of menstruation or duration of stimulation. Discussion This study demonstrated that in patients stimulated with rfsh and GnRH antagonists for IVF, the longer the duration of the menstruation-free interval, the lower the probability of ongoing pregnancy. This was observed in a logistic regression analysis controlling for LH level at initiation of stimulation and for female age, which was also negatively associated with the probability of ongoing pregnancy. All patients analysed in the current study had normal steroid levels at initiation of stimulation, since there is evidence that elevated progesterone levels might be associated with a decreased probability of pregnancy in GnRH antagonist cycles (Kolibianakis et al., 2004b). All patients were treated equally during the follicular phase, as they received the same fixed dose of rfsh and GnRH antagonist was always started on day 6 of stimulation. Moreover, the follicular phase ended in the same way in all patients, since its prolongation has been shown to adversely affect the probability of pregnancy in GnRH antagonist cycles (Kolibianakis et al., 2004a). This was achieved by administering HCG on the first day that 3 follicles of 17 mm were present on ultrasound. Finally, luteal support was the same for all patients. The menstruation-free interval is a parameter that is relevant for the GnRH antagonist and the short agonist protocol. However, it has not so far been investigated and therefore comparison with existing literature is not possible. It is well known that, in the natural cycle, endometrium needs to be stimulated for a period of days after menstruation stops, in order to acquire progesterone receptors that will allow Table IV. Ongoing pregnancy per embryo transfer in groups of patients defined by percentile analysis (thirdile) of the duration of menstruation, duration of stimulation and duration of the menstruation-free interval Ongoing pregnancy n (%) Menstruation duration 0.32 3 4 days 5/28 (10.7) 5 days 8/35 (22.9) 6 days 4/19 (21.1) Duration of stimulation 0.12 7 10 days 12/48 (25.0) 11 days 1/16 (6.3) 12 18 days 2/18 (11.1) Menstruation-free interval 0.02 1 5 days 12/44 (27.3) 6 days 2/15(13.3) 7 days 1/23 (4.3) P a Model P = 0.010; Hosmer and Lemeshow test: P = 0.249. CI = confidence interval. Values in parentheses are percentages. a Exact χ 2 for trend. 1015

E.M.Kolibianakis et al. its luteal transformation following ovulation. In the antagonist cycle, the stimulation of endometrium, by steroids that will allow luteal transformation of endometrium following HCG, starts more abruptly by the end of menstruation. This occurs approximately on stimulation day 4 as a result of the increasing steroid levels. Until that time the menstrual endometrium completes its cycle of re-epithelialization. From that point onward, it appears that there is a period of 5 days during which the endometrium is stimulated by abnormally high E 2 levels, progesterone receptors are induced and preparation for luteal transformation is completed. If this menstruation-free interval is prolonged, the probability of ongoing pregnancy appears to be decreased (Table IV). This can be explained by endometrium abnormalities induced by prolonged hormonal stimulation of endometrium by abnormal E 2 levels. These abnormalities might compromise the probability of pregnancy. Acceptance of this concept would entail analysis of endometrium biopsies taking account of the menstruation-free interval. It would be very interesting if hormonal data in the present study were recorded on the first day after the period had stopped. This would have made it possible to associate not only the duration of the menstruation-free interval with the achievement of ongoing pregnancy, but also the hormonal exposure of endometrium during this period with ongoing pregnancy rates. The possibility that embryo quality, which is crucial for implantation, also affected pregnancy rates in this study is unlikely. The low number of embryos transferred in this population (single embryo transfer in 64.6% of patients), in combination with 13.1 COC retrieved per patient, allowed embryos of high morphological quality to be transferred in all patients. As shown in Table II, morphological embryo quality in this study did not significantly affect the probability of pregnancy. Interestingly, ongoing pregnancy was achieved after a minimum menstruation interval of 1 day, in a 29 year old patient who had six COC retrieved, endometrial thickness of 7 mm on the day of HCG and two top-quality embryos transferred. Although conclusions cannot be drawn on the basis of a single case, it has to be assumed that in this patient endometrium acquired receptivity following HCG administration and progesterone supplementation despite being exposed only 1 day to steroid levels after the end of menstruation. Progesterone receptors, whose stimulation by elevated progesterone level in the luteal phase triggers secretory transformation of endometrium, are known to exist during the early follicular phase in the natural cycle. They are increased by the mid- and late follicular phase, being induced by circulating E 2 levels (Lessey et al., 1988; Bouchard et al., 1991). In this patient, who was exposed only 1 day after the end of menstruation to E 2 stimulation, the progesterone receptor content induced was probably able to allow the endometrium to acquire optimal receptivity for implantation and ongoing pregnancy establishment after the HCG signal. In long protocol GnRH agonist cycles (follicular or luteal), menstruation has ended several days before stimulation with FSH is started. Therefore, the endometrium is subjected to steroids as soon as follicular development begins. However, 1016 although the endometrium in the agonist cycle is exposed earlier to steroids, this occurs more progressively when compared with the antagonist cycle. The estradiol rise is known to be delayed compared to what occurs in the antagonist cycle, since in Phase III comparative trials estradiol levels were significantly higher (almost twice as high) on day 6 of stimulation in the antagonist group, and at the same time more follicles were present in the antagonist than in the agonist group (Borm and Mannaerts, 2000). To date, there have been two histological studies of endometrium prior to HCG administration in IVF cycles using GnRH agonists (Marchini et al., 1991) or GnRH antagonists (Papanikolaou et al., 2005). The studies cannot be compared because of differences in their design but it is interesting to note that histological advancement of endometrium was observed in agonist but not in antagonist cycles prior to HCG administration. In conclusion, the current study provides evidence that prolonged duration of stimulation after menstruation ends is associated with a decreased probability of ongoing pregnancy. 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