Article Aromatase inhibitors in ovarian stimulation for IVF/ICSI: a pilot study

RBMOnline - Vol 13. No 2. 2006 166-172 Reproductive BioMedicine Online; www.rbmonline.com/article/2261 on web 19 May 2006 Article Aromatase inhibitors in ovarian stimulation for IVF/ICSI: a pilot study Willem Verpoest studied medicine, and graduated as an obstetrician gynaecologist at the Catholic University of Louvain, Belgium, in 2003. He developed an interest in reproductive medicine working under Michael Hull and David Cahill in Bristol, UK, and Paul Devroey and Andre Van Steirteghem in Brussels, Belgium, and is currently Fellow at the Centre for Reproductive Medicine at the Free University of Brussels. He is clinical tutor and clinical coordinator for patients undergoing preimplantation genetic diagnosis, one of the main practices at this centre. His main interests are reproductive endocrinology and genetics, and reproductive surgery. Dr Willem Verpoest Willem MJA Verpoest 1,3, Efstratios Kolibianakis 2, Evangelos Papanikolaou 1, Johan Smitz 1, Andre Van Steirteghem 1, Paul Devroey 1 1 Academic Hospital of the Vrije Universiteit Brussel, 101 Laarbeeklaan, B-1090 Brussels, Belgium; 2 Unit for Human Reproduction, 1st Department of Obstetrics and Gynaecology, Aristotle University of Thessaloniki, Greece 3 Correspondence: e-mail: willem.verpoest@az.vub.ac.be; Tel: +32 2 4776699; Fax: +32 2 4776649 Abstract This prospective randomized pilot study was aimed at investigating the effect of the novel addition of aromatase inhibitors to an ovarian stimulation protocol for IVF or intracytoplasmic sperm injection, on endocrine parameters including serum androgen, oestrogen, progesterone, LH and FSH concentrations. The patients were randomized to receiving letrozole (group A; n = 10), versus no letrozole (group B; n = 10) in an ovarian stimulation protocol with recombinant FSH 150 IU/day starting on day 2 of the cycle, and gonadotrophin-releasing hormone antagonist 0.25 mg/day starting on day 6 of the cycle. Median LH concentrations were significantly higher (P P < 0.01) in group A versus group B during letrozole administration. Median serum oestradiol concentrations were lower in group A versus group B, and median serum FSH, testosterone and androstenedione concentrations were higher in group A versus group B, throughout the follicular phase, without reaching significance. Median endometrial thickness was significantly higher (P P < 0.05) in group A versus group B on the day of human chorionic gonadotrophin administration. Pregnancies were achieved. This pilot study supports the idea that aromatase inhibitors can contribute to normal potential of implantation and follicular response, without having negative anti-oestrogenic effects. Keywords: androgens, anti-oestrogens, aromatase inhibitors, ICSI, letrozole, ovarian stimulation 166 Introduction The use of aromatase inhibitors has been shown to be effective in ovulation induction, and appears to be associated with fewer side effects than clomiphene citrate (CC) (Holzer et al., 2006). Moreover, it is associated with improved endometrial thickness, greater follicular response to gonadotrophins and a lower risk of ovarian hyperstimulation syndrome and multiple pregnancies (Metwally and Casper, 2004; Metwally et al., 2005). Aromatase inhibitors such as letrozole are thought to induce the development of a follicle by releasing the pituitary gland from oestrogenic negative feedback, primarily by inhibition of the conversion of androstenedione to oestrone in the ovary. However, there does not appear to be depletion of the central oestrogen receptors, as opposed to what happens when clomiphene citrate is used, thus allowing normal central feedback mechanisms, and therefore monofollicular rather than multifollicular development in most cases (Casper, 2003). Lack of oestrogen receptor depletion in peripheral tissues such as endometrium is the basis for better endometrial development when using aromatase inhibitors (Metwally and Casper, 2001, 2003). Aromatase inhibition is also thought to improve follicular response by augmenting follicular FSH receptor expression as a result of raised intra-ovarian androgen concentrations, although solid evidence for this mechanism is lacking (Metwally and Casper, 2001, 2003). Aromatase inhibition is also thought to improve follicular response by augmenting FSH receptor expression as a result of raised intra-ovarian concentrations, although solid evidence for this mechanism is lacking (Metwally and Casper 2001, 2003; Hugues and Cédrin-Durnerin, 2005). There is little information on the use of aromatase inhibitors in association with

gonadotrophins in ovarian stimulation for IVF/intracytoplasmic sperm injection (ICSI) (Holzer et al., 2006), or on the effect of aromatase inhibitors on serum endocrine parameters, especially on serum androgen concentrations (Metwally et al., 2005). Previous studies have investigated the co-administration, either concomitantly or sequentially, of aromatase inhibitors and gonadotrophins for ovulation induction, and only more recently some limited data emerged on the co-administration of aromatase inhibitors in ovarian stimulation (Metwally et al., 2005). The aim of this pilot study was to investigate the effect of the co-administration of aromatase inhibitors on endocrine parameters, including serum testosterone, androstenedione, oestrogen, progesterone, LH and FSH concentrations, in an ovarian stimulation protocol using recombinant gonadotrophins and gonadotrophin releasing hormone (GnRH) antagonists for IVF/ICSI. Materials and methods This was a prospective, randomized pilot study, at a University Centre for Reproductive Medicine, over an 8-month period from January until September 2003. Inclusion criteria were: (i) subfertility for more than 1 year requiring IVF/ICSI, (ii) age younger than 39 years, (iii) first or second IVF/ICSI trial and (iv) use of ejaculated spermatozoa only. Exclusion criteria were: (i) patients belonging to any of the WHO classification groups (I, II or III) of ovulatory disorders, (ii) oligomenorrhoea (menstrual cycle >35 days), (iii) polymenorrhoea (menstrual cycle<21 days), (iv) early follicular phase FSH concentrations 15 IU/l, (v) endometriosis American Fertility Society (AFS) grades III and IV, (vi) IVF/ICSI with preimplantation genetic diagnosis (PGD) and (vii) body mass index (BMI) 28. Prestudy investigations included: full blood count, renal and liver function tests, thyroid function tests (thyroid-stimulating hormone, thyroxin, antithyroid peroxidase antibodies), prolactin, early follicular phase FSH, LH, oestradiol, progesterone concentrations, and both male and female karyotyping. Transvaginal ultrasound was performed in all patients prior to recruitment. Informed consent was obtained from all patients. The trial was approved by the institutional ethical committee. Patient population Twenty patients with regular ovulation and aged <39 years of age were recruited and randomized according to a computer generated randomization list, 10 in each treatment group. Indications for treatment were similar across the two groups. Ovarian stimulation In group A (n = 10), patients received letrozole (Femara; Novartis Pharma, Basel, Switzerland) 2.5 mg daily from day 2 until day 6 of the cycle and recombinant FSH (rhfsh) (Puregon; NV Organon, Oss, The Netherlands) starting on day 2 of the cycle. In group B (n = 10), ovarian stimulation was performed with rhfsh only, starting on day 2 of the cycle. In both groups, a constant daily dose of 150 IU rhfsh was used for stimulation and GnRH antagonist (ganirelix, Orgalutran; NV Organon) 0.25 mg/day was always started on day 6 of the cycle until the day of ovulation triggering with human chorionic gonadotrophin (HCG). Final oocyte maturation was achieved by the administration of HCG (Pregnyl; Organon) as soon as 3 follicles of 17 mm were present at ultrasound. Steroid concentrations were measured, but were not taken into consideration for the decision on ovulation triggering by HCG administration, which was based exclusively on follicular development. Ultrasound was performed on day 6 of stimulation and thereafter as necessary in order to ensure that HCG was injected on the first day on which 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., 2004). Analysis of serum testosterone, androstenedione, sex hormone binding globulin (SHBG), oestradiol, progesterone, FSH and LH concentrations was performed on days 2, 4, 6, 7, 8 and 10 of the cycle (i.e. days 1, 3, 5, 6, 7 and 9 of the ovarian stimulation). IVF/ICSI procedures The details of the IVF and ICSI procedure have been described previously (Devroey et al., 1995; Joris et al., 1998; Van Steirteghem et al., 1998). The embryo transfer was performed either on day 3 or day 5 after oocyte retrieval. Luteal support consisted of a single dose of HCG 7 days after HCG administration for ovulation triggering. Hormonal and ultrasound assessment Serum LH, FSH, HCG, oestradiol and progesterone were measured with the automated Elecsys immunoanalyser (Roche Diagnostics, Mannheim, Germany). Intra-assay and interassay coefficients of variation (CV) were <3 and <4% for LH, <3 and <6% for FSH, <5 and <7% for HCG, <5 and <10% for oestradiol and <3 and <5% for progesterone respectively. Ultrasound was performed on day 6 of the cycle, plus on the day of HCG, and on other days throughout the stimulation when considered appropriate. Endometrial thickness was measured at the thickest longitudinal plane. Statistical analysis Statistical analysis was carried out using Statistics Package for Social Sciences data editor version 11.0.2. Continuous variables were analysed with the Mann Whitney U-test. - A P-value of <0.05 was considered significant. Variables are expressed as median (range). Results Patient characteristics were similar in the two groups (Table 1). The median number of previous treatment cycles was 0.2 (SD 0.42) in the letrozole + FSH group versus 0.1 (SD 0.33) in the FSH only group. Indications for infertility treatment were similar in both groups (eight oligoasthenoteratozoosperm ia (OAT), one idiopathic and one tubal in the letrozole + FSH group, versus seven OAT, two idiopathic and one tubal in the FSH only group). A non-significant difference in the median age was present between the two groups compared [29.9 (SD 2.78) years in the letrozole + FSH group, versus 32.7 (SD 5.82) 167

Table 1. Patient characteristics and treatment outcome. Group FSH + FSH + letrozole no letrozole No. of patients 10 10 Median age in years (SD) 29.9 (2.78) 32.7 (5.82) Indications Andrological 8 7 Tubal 1 1 Unexplained 1 2 Mean no. of oocytes (SD) 13.8 (9.24) 9.6 (7.73) Fertilization rate % (SD) 63.3 (19.61) 77.84 (18.36) Mean no. of embryos transferred (SD) 1.60 (0.52) 1.60 (0.84) Mean no. of embryos cryopreserved (SD) 2.9 (3.81) 2.55 (4.21) Positive HCG rate per cycle % 50 20 Clinical pregnancy rate per cycle % 50 20 Implantation rate % (ratio) 31.25 (5/10) 12.5 (2/10) The results in the two groups are not statistically different using the Wilcoxon signed rank test. HCG = human chorionic gonadotrophin. in the FSH only group]. The median total dose of recombinant gonadotrophins used per treatment cycle did not differ between the two groups [1575 IU (SD 285.0) in the letrozole + FSH group versus 1650 IU (SD 290.6) in the FSH only group]. Median LH concentrations were significantly higher (P < 0.01) in the letrozole + FSH group versus the FSH only group, on day 4 of the cycle [6.3 IU/l (range 3.7 13.0 IU/l) versus 3.3 IU/l (range 2.0 4.0 IU/l)], and on day 6 of the cycle [2.9 IU/l (range 2.0 4.7 IU/l) versus 1.4 IU/l (range 0.5 3.1 IU/l)] respectively (P < 0.01) (Figure 1). Oestradiol concentrations were lower in the letrozole + FSH group versus the FSH only group (Figure 2), while testosterone (Figure 3), androstenedione (Figure 4) and FSH (Figure 5) concentrations were higher in the letrozole + FSH group versus the FSH only group, throughout the follicular phase, although these differences did not reach significance. Median endometrial thickness was significantly higher in the letrozole + FSH group versus the FSH only group on the day of HCG administration [10.3 mm (minimum 8.2 mm to maximum 15.2 mm) versus 8.1 mm (minimum 6.0 mm to maximum 12.5 mm) respectively; P < 0.05]. A median of 13.8 (SD 9.24) oocytes was retrieved in the letrozole + FSH group, versus 9.6 (SD 7.73) in the FSH only group (not significant, NS, Table 1). The mean fertilization rate in the letrozole + FSH group was 63.3 versus 77.8% in the FSH only group (NS). In both groups, the mean number of embryos transferred was 1.6; the mean number of embryos frozen in the letrozole + FSH group was 2.9, versus 2.55 in the FSH only group (NS). Five ongoing pregnancies were achieved in the letrozole + FSH group, versus two in the FSH only group. 168 Figure 1. Box plots of serum LH concentrations (IU/l). The pale boxes represent median LH concentrations in the letrozole + FSH group (ovarian stimulation with letrozole associated with recombinant FSH); the darker boxes represent median LH concentrations in the FSH only group (ovarian stimulation with recombinant FSH only). Differences between groups were significant at day 4 (P < 0.01), day 6 (P < 0.01) and day 7 of the cycle (P < 0.01).

Figure 2. Box plots of serum oestradiol concentrations (pg/ml). The pale boxes represent median oestradiol concentrations in the letrozole + FSH group (ovarian stimulation with letrozole associated with recombinant FSH); the darker boxes represent oestradiol concentrations in the FSH-only group (ovarian stimulation with recombinant FSH only). Differences were not significant. Figure 3. Box plots of serum testosterone concentrations (μg/l). The pale boxes represent median testosterone concentrations in the letrozole + FSH group (ovarian stimulation with letrozole associated with recombinant FSH); the darker boxes represent median testosterone concentrations in the FSH-only group (ovarian stimulation with recombinant FSH only). Differences were not significant. Figure 4. Box plots of serum androstenedione concentrations (ng/l). The pale boxes represent median androstenedione concentrations in the letrozole + FSH group (ovarian stimulation with letrozole associated with recombinant FSH); the darker boxes represent median androstenedione concentrations in the FSH-only group (ovarian stimulation with recombinant FSH only). Differences were not significant. 169

Figure 5. Box plots of serum FSH concentrations (IU/l). The pale boxes represent serum follicle stimulating hormone (FSH) concentrations in the letrozole + FSH group (ovarian stimulation with letrozole associated with recombinant FSH); the darker boxes represent serum FSH concentrations in the FSH-only group (ovarian stimulation with recombinant FSH only). Differences were not significant. 170 Discussion This study was designed to document the extent to which endocrine parameters change when co-administering aromatase inhibitors in ovarian stimulation for IVF/ICSI, using recombinant gonadotrophins and GnRH antagonists. The results of the pilot study show significantly higher (P < 0.01) LH concentrations, lower oestradiol concentrations, and increased FSH and androgen concentrations, although not significantly so, in the group where the aromatase inhibitor letrozole was added to the ovarian stimulation protocol. An acceptable pregnancy rate was achieved in both groups. Serum oestradiol concentrations (Figure 2) are reduced as a result of the anti-oestrogenic effect of the aromatase inhibitor letrozole, and reflect a decrease of proportionate oestradiol concentrations per follicle. This may limit the negative effect of cumulative oestradiol concentrations on oocyte quality and on endometrial receptivity through modulation of steroid receptors, as proposed by previous authors (Simon et al., 1995; Metwally et al., 2005; Papanikolaou et al., 2005). Moreover, supraphysiological oestrogen concentrations during ovarian stimulation are considered to have a detrimental effect on the developing oocyte, the spermatozoa, the developing embryo and blastocyst hatching, as well as on other targets including leptin, the coagulation system and the early developing placenta (Metwally et al., 2005). These effects are thought to result in reduced implantation rates, reduced pregnancy rates and adverse obstetric outcome following reproductive treatment with ovarian stimulation. Therefore, reducing oestrogen concentrations using aromatase inhibitors, as was the case in the present study, may improve reproductive and obstetric outcome. Nevertheless, the oestradiol concentrations attained during ovarian stimulation with concomitant aromatase inhibitor use are sufficiently high, reflecting adequate follicular development and oestrogen biosynthesis in the presence of anti-oestrogens, as previously demonstrated in primate studies (Weil et al., 1999). Clomiphene citrate has been used in association with ovarian stimulation for IVF/ICSI, but significantly higher concentrations of LH concentrations in the follicular and luteal phase were detected, despite antagonist administration, as well as an increased risk of premature LH surge (Tavaniotou et al., 2002). In the present study, significantly increased LH concentrations (P < 0.01, Figure 1) were also observed, an effect that is only to be expected, due to withdrawal of negative feedback on the pituitary and the hypothalamus. However, the effect of letrozole is short-lived, and should not lead to premature ovulation (Metwally et al., 2005). Indeed, a premature LH surge was not observed in any of the patients. Some authors argue that lower oestradiol concentrations during ovarian stimulation as a result of the use of aromatase inhibition, may even be effective in preventing the occurrence of a premature ovarian LH surge (Metwally et al., 2005). A higher number of oocytes were retrieved in the group receiving letrozole. It is postulated that the observed subtle increase in serum testosterone and androstenedione concentrations in the letrozole group (Figures 3 and 4) may reflect even higher intra-ovarian androgen concentrations, thus contributing to improved folliculogenesis by increasing FSH receptor sensitivity. However, it is still unclear to what extent intrafollicular androgen concentrations change during ovarian stimulation and concomitant aromatase inhibition, and larger studies investigating the effect of aromatase inhibition on androgen metabolism are required (Metwally and Casper, 2001, 2003). This study confirms the findings of other studies, i.e. that the use of aromatase inhibitors is effective in achieving adequate follicular response as well as pregnancies in ovulation induction in both ovulating and anovulating patients (Bulun, 2003; Casper, 2003; Fatemi et al., 2003; Healey et al., 2003). The main action of aromatase inhibitors is through the inhibition of negative oestrogen feedback on the pituitary and

the hypothalamus during administration, thereby allowing production of higher endogenous concentrations of FSH and follicular recruitment and stimulation. The adjunct use of aromatase inhibitors during ovarian stimulation has been shown to improve follicular response and reduce the amount of gonadotrophins required (Kilic-Okman et al., 2003; Metwally and Casper, 2003). The results of the present study, in which a fixed daily dosage of gonadotrophins was used, did not show a difference in the cumulative dosage of gonadotrophin required. The mean number of oocytes retrieved was higher in the letrozole group, which would appear to indicate that the use of aromatase inhibitors might contribute to successful ovarian stimulation with a lower dosage of gonadotrophins. A lower dosage of gonadotrophins used will reduce cost, improve patient compliance and reduce psychological impact (Heijnen et al., 2004; de Klerk et al., 2006), may reduce adverse early obstetric outcome (Metwally et al., 2005) and in the current climate of moving towards a legal restriction of the number of embryos transferred and single embryo transfer (De Neubourg and Gerris, 2003; De Neubourg et al., 2005), a lower and more physiological stimulation is preferred. Lower stimulation may avoid inadequate endometrial synchrony (endometrial advancement) hence improve endometrial receptivity. The proportion of chromosomal abnormalities in embryos may also be reduced as a result of lower stimulation (Baart et al., 2005). The daily 2.5 mg dose of aromatase inhibitor used in this study was adopted from standard adjuvant breast cancer treatment protocols. Other studies have reported both higher dosages (Al-Fozan et al., 2004; Al-Fadhli et al., 2006), and single dose administration (Metwally et al., 2005). A study reporting the results of a randomized trial of 238 cycles of ovulation induction with 7.5 mg of letrozole versus 100 mg of clomiphene citrate daily for 5 days in women with idiopathic infertility did not show a difference in pregnancy rate; however, the miscarriage rate in the clomiphene citrate group was higher (Al-Fozan et al., 2004). A more recent study comparing a daily dose of letrozole of 2.5 mg versus 5 mg showed a higher pregnancy rate and higher follicle count in the latter group (Al-Fadhli et al., 2006). Metwally and colleagues reported the results of a nonrandomized retrospective analysis of single-dose administration of letrozole 20 mg on day 3 of the cycle, showing adequate effectiveness in inducing ovulation in comparison with a regimen of 2.5 mg daily for 5 days (Metwally and Casper, 2005). Further randomized studies are required to define the optimal dose of aromatase inhibitor in ovulation induction and ovarian stimulation. Contrary to the present study where an aromatase inhibitor was used concomitantly with an ovarian stimulation protocol using recombinant gonadotrophins and GnRH antagonists for IVF/ ICSI, other studies have used aromatase inhibitors in sequential rather than concomitant administration. Potential advantages of sequential rather than concomitant administration include a longer time lag until ovulation, allowing clearance from the system, more time for follicular development, and a lower total dose requirement of gonadotrophins (Healey et al., 2003). Other studies into the use of aromatase inhibitors in ovarian stimulation for IVF/ICSI are rare, and have shown potential use in improving ovarian response without provoking any side effects (Metwally et al., 2005). All studies into the use of aromatase inhibitors published so far are of limited size. Recognizing the limitations of the current study with regard to sample size, sources of potential bias were avoided as the study design was randomized controlled, with a fixed dose of recombinant FSH for ovarian stimulation, and a fixed protocol for ovulation triggering as soon as three or more follicles of 17 mm were seen at ultrasound. The current study, as previously stated was a pilot study. It was not possible to perform a meaningful power analysis in the absence of literature data on the co-administration of letrozole and FSH. The present study, as a first, can serve as a source of such information in the design of further randomized controlled trials on the co-administration of aromatase inhibitors and recombinant gonadotrophins. The lack of power to detect a significant difference in certain comparisons performed should not necessarily be interpreted as lack of a true difference between the two groups. In conclusion, the results of this study into the novel coadministration of the aromatase inhibitor letrozole to an ovarian stimulation protocol using recombinant gonadotrophins and GnRH antagonists for IVF/ICSI demonstrate a significant increase (P < 0.01) in LH concentrations, increased serum androgen concentrations and reduced serum oestradiol concentrations in the group receiving the aromatase inhibitor, resulting in an increase in oocytes retrieved and endometrial thickness. Pregnancies were achieved, supporting the idea that aromatase inhibitors can contribute to normal potential of implantation and follicular response, without having negative anti-oestrogenic effects. Larger randomized studies are needed to document the effect of aromatase inhibitors and their endocrine effects on ovarian stimulation for IVF/ICSI and reproductive outcome. Acknowledgement The authors would like to thank Mr Julian Mitchell for his kind linguistic and syntactic assistance. 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