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Combined down-regulation by aromatase inhibitor and GnRH-agonist in IVF patients with endometriomas—A pilot study
European Journal of Obstetrics & Gynecology and Reproductive Biology 144 (2009) 48–53
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European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Combined down-regulation by aromatase inhibitor and GnRH-agonist in IVF patients with endometriomas—A pilot study Kristine Lossl a,*, Anne Loft a, Nina L.C. Freiesleben a, Susanne Bangsbøll a, Claus Yding Andersen b, Anette T. Pedersen c, Dorthe Hartwell c, Anders Nyboe Andersen a a b c
The Fertility Clinic Section 4071, Copenhagen University Hospital, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen, Denmark Laboratory of Reproductive Biology Section 5712, Copenhagen University Hospital, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen, Denmark Department of Obstetrics and Gynaecology, Copenhagen University Hospital, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen, Denmark
A R T I C L E I N F O
A B S T R A C T
Article history: Received 23 September 2008 Received in revised form 29 November 2008 Accepted 1 February 2009
Objective: The key enzyme in the biosynthesis of estradiol, aromatase, has been demonstrated within endometriosis. Combined administration of aromatase inhibitor and GnRH-agonist may efficiently suppress estrogen biosynthesis through a combined pituitary, ovarian, peripheral and ‘‘in situ’’ action. The aim of this study was to test the concept of combined down-regulation prior to IVF in patients with endometriomas. Study design: Prospective pilot study in a university-based tertiary fertility clinic including 20 infertile patients with endometriomas undergoing IVF/ICSI. The patients received goserelin 3.6 mg sc on treatment Days 1, 28 and 56, and one daily tablet of anastrozole 1 mg from Day 1 to Day 69. Controlled ovarian stimulation was initiated from Day 70. Outcome measures were change in endometriomal volume and serum CA125 during down-regulation, standard IVF parameters including pregnancy and delivery rate, and endocrine response. The paired T test and Wilcoxon Signed Rank test were used to analyse paired differences. Results: During the combined down-regulation, the endometriomal volume and the serum CA125 level decreased by 29% (3–39%) and 61% (21–74%), respectively (median (95%CI), P = 0.007 and P = 0.001). In the IVF/ICSI cycle, the number of oocytes retrieved was 7.5 (6.0–10.0) and the fertilization rate was 0.78 (0.38–1.0). Nine patients (45%) conceived, five (25%) had a clinical pregnancy, and three (15%) delivered healthy children (two singletons and one twin). Conclusions: Prolonged combined anastrozole and goserelin down-regulation significantly reduces endometriomal volume and serum CA125, and is compatible with IVF pregnancy and delivery. However, a high pregnancy loss was noted. ß 2009 Elsevier Ireland Ltd. All rights reserved.
Keywords: Aromatase inhibitor Down-regulation Endometrioma GnRH-agonist IVF
1. Introduction Endometriosis is characterized by the presence of endometrial tissue outside the uterus, and infertility is recognized as one of several symptoms related to the disease. Clinical evidence suggests that the IVF pregnancy rate is reduced in women with endometriosis compared to tubal factor controls, and that the IVF pregnancy rate is lower in moderate-severe disease compared to minimal-mild disease [1]. An endometrioma larger than 10 mm classifies a patient as having at least moderate disease [2]. Evidence to support that surgical excision of ovarian endometriomas improves IVF outcome is lacking [3], making research on effective medical inactivation of the disease before IVF highly relevant.
* Corresponding author. E-mail address: [email protected] (K. Lossl). 0301-2115/$ – see front matter ß 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ejogrb.2009.02.001
Aromatase catalyses the final and rate limiting step in the biosynthesis of estrogen and is present in several human tissues including ovaries and adipose tissue. Additionally, aromatase has been demonstrated inside endometriotic tissue indicating an ‘‘in situ’’ synthesis of estradiol [4]. Since endometriosis is an estrogen dependent disease, aromatase inhibitors are obvious candidates for treatment of endometriosis. Third generation aromatase inhibitors are potent and highly selective inhibitors of aromatase [5] and have shown promising results in the treatment of endometriosis related pain and pain recurrence [4]. Due to the marked suppression of estrogen secretion, and reduced pituitary feedback, aromatase inhibitors stimulate gonadotrophin secretion in premenopausal women. This action can be blocked by simultaneous administration of GnRHagonist. Further, prolonged administration of GnRH-agonist before IVF may improve the clinical pregnancy rate in women with endometriosis [6].
K. Lossl et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 144 (2009) 48–53
Our hypothesis is that combined administration of aromatase inhibitor and GnRH-agonist before IVF would maximally inhibit estrogen biosynthesis and endometriosis through combined pituitary, ovarian, peripheral and ‘‘in situ endometriosis’’ action. The primary aim was to study signs of inactivation of the endometriosis during treatment, such as decreased endometriomal volume and serum CA125. Secondary aims were evaluation of the IVF/ICSI cycle including detailed assessment of endocrinology. 2. Materials and methods 2.1. Study design This prospective single-centre study was performed between January 2005 and April 2007. The local Ethics Committee approved the study [Project (KF) 02-073/04]. Each patient gave written informed consent before participating in the study that was conducted according to ‘‘Good Clinical Practice’’ and monitored by an independent external monitor (The GCP-Unit, Copenhagen University Hospital, Gentofte).
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2.4. Ultrasonography Vaginal ultrasound was performed on treatment Days 1, 28, 56, 70 (Stim1), and on the day of hCG by two different physicians, separately, in order to obtain two independent measurements of the endometriomal volume. The mean of the two measures was used in analyses. Each physician measured cyst length and depth in the plane with the largest endometriomal diameter, and the cyst width in the perpendicular plane. The endometriomal volume was calculated using the ellipse formula: D1 D2 D3 0.523. If more than one endometrioma was present, this procedure was repeated, and the endometriomal volume was calculated as the sum of the individual endometriomal volumes. To evaluate follicular response to exogenous gonadotrophin (rFSH) additional scans were performed from stimulation Day 7. A final scan was performed approximately five weeks after embryo transfer to determine endometriomal volume and pregnancy status if pregnant. A ‘‘GE LOGIQ 5 Expert’’ scanner (B-mode, 8.0 MHz) with a transvaginal E8C probe was used.
2.2. Patients 2.5. Blood sampling Twenty-two infertile women meeting the following inclusion criteria and none of the exclusion criteria were enrolled. Inclusion criteria: (i) presence of sonographically diagnosed endometrioma(s) persisting for at least three cycles. An adnexal mass with diffuse low-level internal echoes and no signs of neoplastic features or acute haemorrhage was considered as an endometrioma, (ii) the largest diameter of the largest endometrioma between 20 and 70 mm, (iii) indication for IVF or ICSI, (iv) age 20–39 years, (v) regular menstrual cycle (21–35 days, intra-individual variation 3 days), (vi) BMI 18–30 kg/m2, (vii) negative urinary pregnancy test on treatment Day 1. Exclusion criteria: (i) treatment of endometriosis with GnRH-agonist within the last three months, (ii) history of osteopenia, hepatic, renal, cardiovascular or thomboembolic disorder, and (iii) earlier participation in the trial. One patient was excluded due to inability to follow planned controls; one patient discontinued treatment shortly after Day 28 for private reasons. Twenty patients were treated per protocol and included in analyses. 2.3. Treatment Treatment was initiated (Day 1) on cycle Days 2–4 of a natural cycle (n = 11), or on Day 14+ of an oral contraceptive cycle (n = 9), enabling exclusion of pregnancy before administration of investigational drugs. One daily tablet of anastrozole 1 mg (Arimidex, AstraZeneca AS, Denmark) was administered from Day 1 to Day 69. Goserelin 3.6 mg sc (Zoladex, AstraZeneca AS, Denmark) was administered on Days 1, 28 and 56. Controlled ovarian stimulation (COS) was initiated on Day 70 using an individual dose of recombinant FSH (Gonal f, Serono Europe Limited, United Kingdom). In one case COS was postponed 14 days because of flare-up with functional cysts after the 3rd injection of goserelin. When minimum two follicles reached a mean diameter of 17 mm, 250 mg recombinant hCG (Ovitrelle, Serono Europe Limited, United Kingdom) was administered followed by oocyte retrieval 36 h later. IVF (n = 16) or ICSI (n = 4) was performed, and one or two embryos were transferred on Day 2. Embryos with >20% fragmentation and/or multinucleated blastomer(s) were not transferred. Vaginal progesterone was administered from day of embryo transfer and two weeks onwards.
Serum concentrations of FSH, LH, progesterone, testosterone, estradiol and CA125 were measured on Days 1, 28, 56, 70/Stim1, Stim7, day of hCG and approximately five weeks after embryo transfer. Analyses were performed by electrochemiluminescence immunoassay (Roche Diagnostics, Mannheim, Germany) in Modular analytics E170 immunoassay analyser. Inter-assay coefficients of variation were 4.0% for FSH, 2.1% for LH, 4.8% for progesterone, 6.0% for testosterone and 4.9% for estradiol. 2.6. Study endpoints Primary endpoints were changes in the endometriomal volume and serum CA125 from Day 1 to Day 70. Secondary endpoints were standard IVF/ICSI parameters and the endocrine responses during treatment. 2.7. Statistical analysis Data are presented by the median with corresponding range or 95% confidence interval (CI) unless otherwise is specified. The paired T test and Wilcoxon Signed Rank test were used to analyse paired differences depending on whether or not these were compatible with a normal distribution. Agreement between the two separate measurements of the endometriomal volume was assessed by calculation of the mean difference and limits of agreement. The software package SPSS, version 14 (SPSS Inc., Chicago, USA) was used for analyses. A two-sided P value <0.05 was considered statistically significant. 3. Results 3.1. Patient characteristics Demographics and clinical characteristics are shown in Table 1. More than one endometrioma was present in 9/20 patients, and 5/20 had bilateral endometriomas. Besides the ultrasonic diagnosis of endometrioma(s) that had persisted for at least three cycles, 11/20 had been operated for endometriosis in the past.
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K. Lossl et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 144 (2009) 48–53
Table 1 Baseline characteristics (n = 20).
Table 2 Stimulation characteristics and cycle outcome (n = 20).
Age BMI
31.0 (26.0–37.0) 23.1 (18.0–29.7)
Days of stimulation Total units of rFSH
10 (8–17) 2512.5 (1350–6600)
Infertility diagnosis [n (%)] Endometriosis only Endometriosis and tubal factor Endometriosis and male factor
11 (55) 4 (20) 5 (25)
Day of hCG Endometrium (mm) Follicles > 10 mm (mean SD) Follicles > 14 mm (mean SD)
8.6 (5.3–13.5) 11 (4–25) (11.6 5.2) 8 (4–13) (8.6 2.4)
Endometriomas One endometrioma present [n (%)] Two endometriomas present [n (%)] Three endometriomas present [n (%)] Unilateral endometrioma(s) [n (%)] Bilateral endometriomas [n (%)]
11 7 2 15 5
Endometriomal volume Day 1 (ml) CA 125 Day 1 (U/ml)a Oral contraceptive pills on Day 1 [n (%)]b Years since diagnosis of endometriosis Endometriosis operation in the past [n (%)] Earlier IVF/ICSI treatments
21.3 (3.8–78.8) 57.5 (8.0–173.0) 9 (45) 1 (0.3–7.0) 11 (55) 1 (0–5)
Oocytes retrieved per pick-up ICSI as fertilization method [n (%)] Fertilization ratea (mean SD) Embryos per pick-up (mean SD) Patients reaching embryo transfer (n) Embryos transferred Patients having embryos cryopreserved (n) Embryos cryopreserved (mean SD)
7.5 (2–12) (7.7 3.0) 4 (20) 0.78 (0–1) (0.6 7 0.34) 3 (0–10) (4.3 2.9) 19 2 (1–2) 10 3.5 (1–7) (3.5 2.0)
Positive hCGb [n (% per initiated cycle)] Biochemichal pregnancy Early spontaneous abortion Delivery
9 (45) 4 2 3
Implantation rate % Ongoing implantation rate % Clinical pregnancyc rate [n (% per initiated cycle)] Live birthsd [n (% per initiated cycle)]
25 (8/32) 13 (4/32) 5 (25) 3 (15)
(55) (35) (10) (75) (25)
Numerical data are presented as median with the range shown in brackets. a Levels of FSH, LH, estradiol, testosterone and progesterone are shown in Table 4. b Oral contraceptive pills were used to plan treatment start in nine patients with a planned 5-day treatment overlap.
3.2. Endometriomal volume and CA125 during treatment The changes in endometriomal volume and serum CA125 are shown in Fig. 1. Fifteen patients (75%) experienced a reduction of cyst volume from Day 1 to Day 70, being >25% in 13 patients. No change was recorded in two patients (10%), and three patients (15%) experienced an increase of the endometriomal volume, being >25% in two patients. The median change in endometriomal volume from Day 1 to Day 70 was minus 29% (95%CI: 3–39%, P = 0.007). The CA125 level was reduced by 61% (95%CI: 21–74%, P = 0.001) from Day 1 to Day 70, and by 36% (95%CI: 17–52%, P = 0.001) from Day 28 to Day 70. Eleven women had menstrual bleeding on Day 1, whereas no women were bleeding on Days 28 and 70. The proportion of patients with serum CA125 above normal level (>35 U/ml) was 85% (17/20) before treatment (Day 1), 55% (11/20) on treatment Day 28, 30% (6/20) on Day 56, and 25% (5/20) on Day 70. After treatment the endometriomal volume (P = 0.028) and the CA125 level (P < 0.001) increased significantly from day of hCG until five weeks after embryo transfer, Fig. 1.
Data are presented as median (range) unless otherwise is specified. a Fertilized oocytes divided by oocytes retrieved (IVF) or oocytes injected (ICSI). One patient had total fertilization failure after IVF. b Serum beta-hCG >2 IU/l two weeks after embryo transfer. c Appearance of gestational sac(s). d Two singleton and one twin delivery.
Two independent investigators measured the total endometriomal volume of the 20 patients on Days 1, 28, 56, 70, and on the day of hCG, giving a total of 100 paired measurements. The mean difference in measurements was 0.31 ml with 95% of the differences being in the interval 6.61 to 7.23 ml (limits of agreement). 3.3. Characteristics of the IVF/ICSI cycle following the prolonged combined down-regulation Table 2 shows the stimulation characteristics and cycle outcome of the IVF/ICSI treatment following the prolonged combined down-regulation. When key characteristics of the IVF/ICSI treatment following the prolonged combined down-regulation were compared to a standard IVF/ICSI treatment in 15 women who underwent a
Fig. 1. Endometriomal volume and CA125 during treatment. Day 1 = Treatment Day 1, where the first Zoladex 3.6 mg sc was administered, and daily intake of Arimidex 1 mg was initiated. The second and third Zoladex 3.6 mg was administered on Day 28 and Day 56, respectively. Day 70 corresponds to stimulation Day 1, where ovarian stimulation was initiated with rFSH and daily intake of Arimidex was stopped.
K. Lossl et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 144 (2009) 48–53 Table 3 Key IVF/ICSI characteristics following prolonged combined down-regulationa or standard treatmentb in those patients who had a previous or subsequent standard cycle with the endometrioma(s) being present (n = 15).
Days of stimulation Total units of rFSH Oocytes retrieved per pick-up Fertilization rate Embryos per pick-up (n)
Study cyclea
Standard cycleb
P-value
10 (8–17) 2725 (1500–6600) 7 (2–12) 0.75 (0–1) 2 (0–8)
11 (7–13) 2600 (1538–5400) 8 (2–14) 0.75 (0–1) 4 (0–9)
0.521 0.500 0.578 0.715 0.189
Data are presented as median (range). Paired samples T test was used to compare the Study cycle to the Standard cycle. aStudy cycle refers to the investigated IVF/ICSI cycle that immediately followed prolonged combined aromatase inhibitor and GnRHagonist down-regulation. bIf the women had undergone previous standard IVF/ICSI treatment(s), the one just preceding the Study cycle was chosen (n = 9). If no previous standard treatments, the first following standard cycle was chosen (n = 6).
previous or subsequent standard cycle, with the endometrioma(s) being present, there were no significant differences in number of stimulation days, total FSH dose, oocytes retrieved, fertilization rate or number of embryos, Table 3. 3.4. Endocrinology during the combined down-regulation Serum levels of FSH, LH, progesterone, testosterone and estradiol are shown in Table 4. The level of LH (P = 0.016) and estradiol (P = 0.001) decreased significantly from Day 1 to Day 70, in spite of reduced levels on Day 1, as 9 women took oral contraceptive pills. Among 11 women initiating treatment in their natural cycle, FSH decreased significantly from 6.5 IU/l (95%CI: 5.6–10.5) on Day 1 to 4.7 IU/l (95%CI: 2.8–5.9) on Day 70 (P = 0.007). The testosterone and progesterone levels did not change significantly during the combined down-regulation. 3.5. Adverse reactions/events No serious adverse reactions or events occurred. Sixteen women (80%) had vaginal spotting/bleeding after the first injection of Zoladex 3.6 mg sc, indicating some degree of flare-up. Two women (10%) had flare-up after the second Zoladex, and one woman (5%) after the third Zoladex, with functional ovarian cysts visible by ultrasound. 4. Discussion To our knowledge this is the first time prolonged combined down-regulation by aromatase inhibitor and GnRH-agonist has been investigated in infertile women with endometriomas/endometriosis undergoing IVF. The volume of the endometriomas and the serum level of CA125 decreased significantly during the treatment indicating inactivation of the disease. Further, it was demonstrated that prolonged combined down-regulation, including daily admin-
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istration of anastrozole, was compatible with IVF pregnancy and the delivery of healthy children following COS. Contradictory results have been published regarding the effect of prolonged GnRH-agonist administration on the size of endometriomas. In some studies the cyst size was not affected [7,8], whereas a reduction after six months of treatment was reported by others [9–11]. Regarding serum CA125, administration of GnRH-agonist for three months significantly reduced levels in patients with moderate and severe endometriosis to 24 U/ml (SD 10) and 26 U/ml (SD 12), respectively [12]. This seems quite similar to the level found after 2.5 months of combined down-regulation in the present study (median 20.5 U/ml, range 10–65). The serum CA125 level is higher during menstruation compared to other parts of the menstrual cycle, especially in women with endometriosis [13]. In the present study, 11 of 20 patients initiated treatment during their menstruation (Day 1), which contributed to elevated CA125 levels measured on Day 1 (Fig. 1). It is therefore important to notice that the CA125 level not only decreased significantly from treatment Day 1 to Day 70, but also from Day 28, where no women had vaginal bleeding, until Day 70. The present study documents the effect of combined aromatase inhibitor and GnRH-agonist to shrink endometriomas and reduce CA125, but an additional effect of adding the aromatase inhibitor, compared to the classical GnRH-agonist alone regiment, remains unproven. Only one randomized controlled trial was published on combined administration of aromatase inhibitor and GnRHagonist [14]. Following surgery for severe endometriosis, six months of anastrozole and goserelin treatment increased the painfree interval and decreased symptom recurrence rates compared to goserelin alone indicating an additional effect of the aromatase inhibitor, but objective parameters, such as the size of endometriomas, has not been recorded in a randomized setting. We therefore still need sufficiently powered randomized trials comparing this new approach to the usual GnRH-agonist treatment. Regarding IVF cycle outcome, a mean fertilization rate of 69.9%, an implantation rate of 14.1%, and a clinical pregnancy rate of 22.7%, were recently reported among 56 women with ultrasounddiagnosed endometriomas >3 cm who followed a standard long agonist protocol [15]. Although several differences exist between this study and the present study, the referred results are remarkable similar. Further, when the IVF/ICSI cycle including prolonged combined down-regulation was compared to a standard IVF/ICSI cycle in 15 patients who served as their own controls, neither the number of oocytes retrieved, the fertilization rate, nor the number of embryos was improved. The present data, therefore, do not prove that prolonged combined aromatase inhibitor and GnRH-agonist down-regulation improves IVF outcome. Interestingly, 9 of the 20 patients (45%) conceived, but 6 of these pregnancies were biochemical pregnancies (n = 4) or early
Table 4 Serum endocrinology during treatment (n = 20). Day 1a
FSH (IU/l) LH (IU/l) Progesterone (nmol/l) Testosterone (nmol/l) Estradiol (nmol/l)
4.5 (0.9–6.5) 2.7 (0.2–5.1) 1.5 (1.0–2.3) 1.1 (0.8–1.7) 0.12 (0.05–0.15)
Day 28
3.7 (2.1–6.3) 0.5 (0.2–0.7) 1.5 (1.1–1.8) 1.3 (0.8–1.5) 0.04 (0.04–0.05)
Day 56
4.9 (3.6–6.2) 0.3 (0.2–0.4) 1.3 (1.1–1.6) 1.0 (0.6–1.3) 0.04 (0.04–0.05)
Day 70
Day hCG
Stim 1
Stim 7
4.8 (3.0–5.7) 0.6 (0.4–0.9) 1.5 (1.1–2.0) 1.1 (0.8–1.4) 0.04 (0.04–0.06)
14.7 (9.8–16.6) 0.4 (0.3–0.6) 1.7 (1.3–2.0) 1.3 (0.8–1.6) 0.49 (0.23–1.13)
15.1 (13.7–20.3) 0.5 (0.3–0.9) 2.2 (1.6–2.9) 1.4 (0.9–1.8) 2.92 (1.61–5.06)
Data are presented as median (95%CI). a On Day 1 (treatment Day 1), nine patients (45%) had taken oral contraceptive pills during at least 14 days to control treatment start. Among the 11 patients that began treatment in their natural menstrual cycle (Days 2–4) the hormonal levels on Day 1 were: FSH 6.5 (5.6–10.5), LH 5.1 (3.2–10.0), progesterone 1.5 (1.0–3.7), testosterone 1.3 (0.7–2.1) and estradiol 0.15 (0.12–0.17).
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spontaneous abortions (n = 2), and only 3 patients (15%) reached delivery. This could be accidental, but an impaired quality of the oocytes and/or the endometrium following prolonged combined down-regulation must be considered. Short-term use of aromatase inhibitor before ovarian stimulation increases the androgen level in preovulatory follicular fluid [16,17]. Further, very low serum estradiol and LH levels were noted during the combined downregulation, Table 4. It has earlier been suggested that the estradiol to androgen ratio was higher in follicles containing an oocyte with a better developmental potential [18,19]. The present protocol may therefore benefit from withdrawal of the aromatase inhibitor one week before initiating exogenous gonadotrophin stimulation allowing a ‘‘washout period’’ securing clearance of the aromatase inhibitor with an half-life of 40 h (anastrozole) [20]. Low endogenous LH levels and lack of exogenous LH support have been implicated in less high-quality embryos for cryopreservation [21], lower pregnancy rate [22,23], and an increased miscarriage rate [24]. Limited literature exists on prolonged GnRHagonist down-regulation followed by rFSH stimulation. A high cumulative IVF pregnancy rate after six months of goserelin downregulation and ovarian stimulation with rFSH has been reported [25], but it was not reported in which cycle number the pregnancies occurred leaving it unknown how many occurred directly after the prolonged agonist down-regulation. It might have been beneficial to use hMG or rLH supplementation during the ovarian stimulation in the present study. Several unexpected incidences of flare-up were noted after the second and third administration of goserelin 3.6 mg. The profound suppression of estradiol during the combined treatment may activate pituitary FSH secretion to a higher extent than treatment with the agonist alone. One possibility to overcome this could be reduced intervals between goserelin administrations. Based on the issues discussed above, we propose an experimental protocol in infertile endometriosis patients with shortened intervals between depot GnRH-agonist administrations, and cessation of the aromatase inhibitor one week before COS by hMG. This experimental protocol should be tested in a randomized study design including a control group receiving only the depot GnRH-agonist. In the present study the diagnosis of endometrioma was made by transvaginal ultrasound by two independent physicians. Several investigations have demonstrated the validity of ultrasound to diagnose endometriomas [26,27]. Further, the endometriomas had persisted though at least three months, also in patients with a previous histopathological diagnosis of endometriosis, to reduce the risk of misdiagnosing a haemorrhagic cyst as an endometrioma. In conclusion, 2.5 months of combined aromatase inhibitor and GnRH-agonist administration significantly decreased the volume of endometriomas and the serum level of CA125 in infertile women with endometriomas. IVF following the combined down-regulation was compatible with pregnancy and delivery of healthy children, but a high pregnancy loss was noted. Possible enhanced endometriosis inactivation and signs of improved IVF cycle outcome compared to down-regulation by the agonist alone need to be evaluated in a controlled study design. Conflict of interest None. Acknowledgements The authors thank Dr. J. Ingemanssen and Dr. A. Lindhard for their help in recruiting patients. Merck Serono is thanked for an
unrestricted educational grant supporting Kristine Lossl. AstraZeneca is thanked for covering the costs of Zoladex.
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logs in patients with endometriosis. Fertil Steril 2002;78(October (4)):757– 62. [26] Moore J, Copley S, Morris J, Lindsell D, Golding S, Kennedy S. A systematic review of the accuracy of ultrasound in the diagnosis of endometriosis. Ultrasound Obstet Gynecol 2002;20(December (6)): 630–4. [27] Patel MD, Feldstein VA, Chen DC, Lipson SD, Filly RA. Endometriomas: diagnostic performance of US. Radiology 1999;210(March (3)):739–45.
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European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Combined down-regulation by aromatase inhibitor and GnRH-agonist in IVF patients with endometriomas—A pilot study Kristine Lossl a,*, Anne Loft a, Nina L.C. Freiesleben a, Susanne Bangsbøll a, Claus Yding Andersen b, Anette T. Pedersen c, Dorthe Hartwell c, Anders Nyboe Andersen a a b c
The Fertility Clinic Section 4071, Copenhagen University Hospital, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen, Denmark Laboratory of Reproductive Biology Section 5712, Copenhagen University Hospital, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen, Denmark Department of Obstetrics and Gynaecology, Copenhagen University Hospital, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen, Denmark
A R T I C L E I N F O
A B S T R A C T
Article history: Received 23 September 2008 Received in revised form 29 November 2008 Accepted 1 February 2009
Objective: The key enzyme in the biosynthesis of estradiol, aromatase, has been demonstrated within endometriosis. Combined administration of aromatase inhibitor and GnRH-agonist may efficiently suppress estrogen biosynthesis through a combined pituitary, ovarian, peripheral and ‘‘in situ’’ action. The aim of this study was to test the concept of combined down-regulation prior to IVF in patients with endometriomas. Study design: Prospective pilot study in a university-based tertiary fertility clinic including 20 infertile patients with endometriomas undergoing IVF/ICSI. The patients received goserelin 3.6 mg sc on treatment Days 1, 28 and 56, and one daily tablet of anastrozole 1 mg from Day 1 to Day 69. Controlled ovarian stimulation was initiated from Day 70. Outcome measures were change in endometriomal volume and serum CA125 during down-regulation, standard IVF parameters including pregnancy and delivery rate, and endocrine response. The paired T test and Wilcoxon Signed Rank test were used to analyse paired differences. Results: During the combined down-regulation, the endometriomal volume and the serum CA125 level decreased by 29% (3–39%) and 61% (21–74%), respectively (median (95%CI), P = 0.007 and P = 0.001). In the IVF/ICSI cycle, the number of oocytes retrieved was 7.5 (6.0–10.0) and the fertilization rate was 0.78 (0.38–1.0). Nine patients (45%) conceived, five (25%) had a clinical pregnancy, and three (15%) delivered healthy children (two singletons and one twin). Conclusions: Prolonged combined anastrozole and goserelin down-regulation significantly reduces endometriomal volume and serum CA125, and is compatible with IVF pregnancy and delivery. However, a high pregnancy loss was noted. ß 2009 Elsevier Ireland Ltd. All rights reserved.
Keywords: Aromatase inhibitor Down-regulation Endometrioma GnRH-agonist IVF
1. Introduction Endometriosis is characterized by the presence of endometrial tissue outside the uterus, and infertility is recognized as one of several symptoms related to the disease. Clinical evidence suggests that the IVF pregnancy rate is reduced in women with endometriosis compared to tubal factor controls, and that the IVF pregnancy rate is lower in moderate-severe disease compared to minimal-mild disease [1]. An endometrioma larger than 10 mm classifies a patient as having at least moderate disease [2]. Evidence to support that surgical excision of ovarian endometriomas improves IVF outcome is lacking [3], making research on effective medical inactivation of the disease before IVF highly relevant.
* Corresponding author. E-mail address: [email protected] (K. Lossl). 0301-2115/$ – see front matter ß 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ejogrb.2009.02.001
Aromatase catalyses the final and rate limiting step in the biosynthesis of estrogen and is present in several human tissues including ovaries and adipose tissue. Additionally, aromatase has been demonstrated inside endometriotic tissue indicating an ‘‘in situ’’ synthesis of estradiol [4]. Since endometriosis is an estrogen dependent disease, aromatase inhibitors are obvious candidates for treatment of endometriosis. Third generation aromatase inhibitors are potent and highly selective inhibitors of aromatase [5] and have shown promising results in the treatment of endometriosis related pain and pain recurrence [4]. Due to the marked suppression of estrogen secretion, and reduced pituitary feedback, aromatase inhibitors stimulate gonadotrophin secretion in premenopausal women. This action can be blocked by simultaneous administration of GnRHagonist. Further, prolonged administration of GnRH-agonist before IVF may improve the clinical pregnancy rate in women with endometriosis [6].
K. Lossl et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 144 (2009) 48–53
Our hypothesis is that combined administration of aromatase inhibitor and GnRH-agonist before IVF would maximally inhibit estrogen biosynthesis and endometriosis through combined pituitary, ovarian, peripheral and ‘‘in situ endometriosis’’ action. The primary aim was to study signs of inactivation of the endometriosis during treatment, such as decreased endometriomal volume and serum CA125. Secondary aims were evaluation of the IVF/ICSI cycle including detailed assessment of endocrinology. 2. Materials and methods 2.1. Study design This prospective single-centre study was performed between January 2005 and April 2007. The local Ethics Committee approved the study [Project (KF) 02-073/04]. Each patient gave written informed consent before participating in the study that was conducted according to ‘‘Good Clinical Practice’’ and monitored by an independent external monitor (The GCP-Unit, Copenhagen University Hospital, Gentofte).
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2.4. Ultrasonography Vaginal ultrasound was performed on treatment Days 1, 28, 56, 70 (Stim1), and on the day of hCG by two different physicians, separately, in order to obtain two independent measurements of the endometriomal volume. The mean of the two measures was used in analyses. Each physician measured cyst length and depth in the plane with the largest endometriomal diameter, and the cyst width in the perpendicular plane. The endometriomal volume was calculated using the ellipse formula: D1 D2 D3 0.523. If more than one endometrioma was present, this procedure was repeated, and the endometriomal volume was calculated as the sum of the individual endometriomal volumes. To evaluate follicular response to exogenous gonadotrophin (rFSH) additional scans were performed from stimulation Day 7. A final scan was performed approximately five weeks after embryo transfer to determine endometriomal volume and pregnancy status if pregnant. A ‘‘GE LOGIQ 5 Expert’’ scanner (B-mode, 8.0 MHz) with a transvaginal E8C probe was used.
2.2. Patients 2.5. Blood sampling Twenty-two infertile women meeting the following inclusion criteria and none of the exclusion criteria were enrolled. Inclusion criteria: (i) presence of sonographically diagnosed endometrioma(s) persisting for at least three cycles. An adnexal mass with diffuse low-level internal echoes and no signs of neoplastic features or acute haemorrhage was considered as an endometrioma, (ii) the largest diameter of the largest endometrioma between 20 and 70 mm, (iii) indication for IVF or ICSI, (iv) age 20–39 years, (v) regular menstrual cycle (21–35 days, intra-individual variation 3 days), (vi) BMI 18–30 kg/m2, (vii) negative urinary pregnancy test on treatment Day 1. Exclusion criteria: (i) treatment of endometriosis with GnRH-agonist within the last three months, (ii) history of osteopenia, hepatic, renal, cardiovascular or thomboembolic disorder, and (iii) earlier participation in the trial. One patient was excluded due to inability to follow planned controls; one patient discontinued treatment shortly after Day 28 for private reasons. Twenty patients were treated per protocol and included in analyses. 2.3. Treatment Treatment was initiated (Day 1) on cycle Days 2–4 of a natural cycle (n = 11), or on Day 14+ of an oral contraceptive cycle (n = 9), enabling exclusion of pregnancy before administration of investigational drugs. One daily tablet of anastrozole 1 mg (Arimidex, AstraZeneca AS, Denmark) was administered from Day 1 to Day 69. Goserelin 3.6 mg sc (Zoladex, AstraZeneca AS, Denmark) was administered on Days 1, 28 and 56. Controlled ovarian stimulation (COS) was initiated on Day 70 using an individual dose of recombinant FSH (Gonal f, Serono Europe Limited, United Kingdom). In one case COS was postponed 14 days because of flare-up with functional cysts after the 3rd injection of goserelin. When minimum two follicles reached a mean diameter of 17 mm, 250 mg recombinant hCG (Ovitrelle, Serono Europe Limited, United Kingdom) was administered followed by oocyte retrieval 36 h later. IVF (n = 16) or ICSI (n = 4) was performed, and one or two embryos were transferred on Day 2. Embryos with >20% fragmentation and/or multinucleated blastomer(s) were not transferred. Vaginal progesterone was administered from day of embryo transfer and two weeks onwards.
Serum concentrations of FSH, LH, progesterone, testosterone, estradiol and CA125 were measured on Days 1, 28, 56, 70/Stim1, Stim7, day of hCG and approximately five weeks after embryo transfer. Analyses were performed by electrochemiluminescence immunoassay (Roche Diagnostics, Mannheim, Germany) in Modular analytics E170 immunoassay analyser. Inter-assay coefficients of variation were 4.0% for FSH, 2.1% for LH, 4.8% for progesterone, 6.0% for testosterone and 4.9% for estradiol. 2.6. Study endpoints Primary endpoints were changes in the endometriomal volume and serum CA125 from Day 1 to Day 70. Secondary endpoints were standard IVF/ICSI parameters and the endocrine responses during treatment. 2.7. Statistical analysis Data are presented by the median with corresponding range or 95% confidence interval (CI) unless otherwise is specified. The paired T test and Wilcoxon Signed Rank test were used to analyse paired differences depending on whether or not these were compatible with a normal distribution. Agreement between the two separate measurements of the endometriomal volume was assessed by calculation of the mean difference and limits of agreement. The software package SPSS, version 14 (SPSS Inc., Chicago, USA) was used for analyses. A two-sided P value <0.05 was considered statistically significant. 3. Results 3.1. Patient characteristics Demographics and clinical characteristics are shown in Table 1. More than one endometrioma was present in 9/20 patients, and 5/20 had bilateral endometriomas. Besides the ultrasonic diagnosis of endometrioma(s) that had persisted for at least three cycles, 11/20 had been operated for endometriosis in the past.
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K. Lossl et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 144 (2009) 48–53
Table 1 Baseline characteristics (n = 20).
Table 2 Stimulation characteristics and cycle outcome (n = 20).
Age BMI
31.0 (26.0–37.0) 23.1 (18.0–29.7)
Days of stimulation Total units of rFSH
10 (8–17) 2512.5 (1350–6600)
Infertility diagnosis [n (%)] Endometriosis only Endometriosis and tubal factor Endometriosis and male factor
11 (55) 4 (20) 5 (25)
Day of hCG Endometrium (mm) Follicles > 10 mm (mean SD) Follicles > 14 mm (mean SD)
8.6 (5.3–13.5) 11 (4–25) (11.6 5.2) 8 (4–13) (8.6 2.4)
Endometriomas One endometrioma present [n (%)] Two endometriomas present [n (%)] Three endometriomas present [n (%)] Unilateral endometrioma(s) [n (%)] Bilateral endometriomas [n (%)]
11 7 2 15 5
Endometriomal volume Day 1 (ml) CA 125 Day 1 (U/ml)a Oral contraceptive pills on Day 1 [n (%)]b Years since diagnosis of endometriosis Endometriosis operation in the past [n (%)] Earlier IVF/ICSI treatments
21.3 (3.8–78.8) 57.5 (8.0–173.0) 9 (45) 1 (0.3–7.0) 11 (55) 1 (0–5)
Oocytes retrieved per pick-up ICSI as fertilization method [n (%)] Fertilization ratea (mean SD) Embryos per pick-up (mean SD) Patients reaching embryo transfer (n) Embryos transferred Patients having embryos cryopreserved (n) Embryos cryopreserved (mean SD)
7.5 (2–12) (7.7 3.0) 4 (20) 0.78 (0–1) (0.6 7 0.34) 3 (0–10) (4.3 2.9) 19 2 (1–2) 10 3.5 (1–7) (3.5 2.0)
Positive hCGb [n (% per initiated cycle)] Biochemichal pregnancy Early spontaneous abortion Delivery
9 (45) 4 2 3
Implantation rate % Ongoing implantation rate % Clinical pregnancyc rate [n (% per initiated cycle)] Live birthsd [n (% per initiated cycle)]
25 (8/32) 13 (4/32) 5 (25) 3 (15)
(55) (35) (10) (75) (25)
Numerical data are presented as median with the range shown in brackets. a Levels of FSH, LH, estradiol, testosterone and progesterone are shown in Table 4. b Oral contraceptive pills were used to plan treatment start in nine patients with a planned 5-day treatment overlap.
3.2. Endometriomal volume and CA125 during treatment The changes in endometriomal volume and serum CA125 are shown in Fig. 1. Fifteen patients (75%) experienced a reduction of cyst volume from Day 1 to Day 70, being >25% in 13 patients. No change was recorded in two patients (10%), and three patients (15%) experienced an increase of the endometriomal volume, being >25% in two patients. The median change in endometriomal volume from Day 1 to Day 70 was minus 29% (95%CI: 3–39%, P = 0.007). The CA125 level was reduced by 61% (95%CI: 21–74%, P = 0.001) from Day 1 to Day 70, and by 36% (95%CI: 17–52%, P = 0.001) from Day 28 to Day 70. Eleven women had menstrual bleeding on Day 1, whereas no women were bleeding on Days 28 and 70. The proportion of patients with serum CA125 above normal level (>35 U/ml) was 85% (17/20) before treatment (Day 1), 55% (11/20) on treatment Day 28, 30% (6/20) on Day 56, and 25% (5/20) on Day 70. After treatment the endometriomal volume (P = 0.028) and the CA125 level (P < 0.001) increased significantly from day of hCG until five weeks after embryo transfer, Fig. 1.
Data are presented as median (range) unless otherwise is specified. a Fertilized oocytes divided by oocytes retrieved (IVF) or oocytes injected (ICSI). One patient had total fertilization failure after IVF. b Serum beta-hCG >2 IU/l two weeks after embryo transfer. c Appearance of gestational sac(s). d Two singleton and one twin delivery.
Two independent investigators measured the total endometriomal volume of the 20 patients on Days 1, 28, 56, 70, and on the day of hCG, giving a total of 100 paired measurements. The mean difference in measurements was 0.31 ml with 95% of the differences being in the interval 6.61 to 7.23 ml (limits of agreement). 3.3. Characteristics of the IVF/ICSI cycle following the prolonged combined down-regulation Table 2 shows the stimulation characteristics and cycle outcome of the IVF/ICSI treatment following the prolonged combined down-regulation. When key characteristics of the IVF/ICSI treatment following the prolonged combined down-regulation were compared to a standard IVF/ICSI treatment in 15 women who underwent a
Fig. 1. Endometriomal volume and CA125 during treatment. Day 1 = Treatment Day 1, where the first Zoladex 3.6 mg sc was administered, and daily intake of Arimidex 1 mg was initiated. The second and third Zoladex 3.6 mg was administered on Day 28 and Day 56, respectively. Day 70 corresponds to stimulation Day 1, where ovarian stimulation was initiated with rFSH and daily intake of Arimidex was stopped.
K. Lossl et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 144 (2009) 48–53 Table 3 Key IVF/ICSI characteristics following prolonged combined down-regulationa or standard treatmentb in those patients who had a previous or subsequent standard cycle with the endometrioma(s) being present (n = 15).
Days of stimulation Total units of rFSH Oocytes retrieved per pick-up Fertilization rate Embryos per pick-up (n)
Study cyclea
Standard cycleb
P-value
10 (8–17) 2725 (1500–6600) 7 (2–12) 0.75 (0–1) 2 (0–8)
11 (7–13) 2600 (1538–5400) 8 (2–14) 0.75 (0–1) 4 (0–9)
0.521 0.500 0.578 0.715 0.189
Data are presented as median (range). Paired samples T test was used to compare the Study cycle to the Standard cycle. aStudy cycle refers to the investigated IVF/ICSI cycle that immediately followed prolonged combined aromatase inhibitor and GnRHagonist down-regulation. bIf the women had undergone previous standard IVF/ICSI treatment(s), the one just preceding the Study cycle was chosen (n = 9). If no previous standard treatments, the first following standard cycle was chosen (n = 6).
previous or subsequent standard cycle, with the endometrioma(s) being present, there were no significant differences in number of stimulation days, total FSH dose, oocytes retrieved, fertilization rate or number of embryos, Table 3. 3.4. Endocrinology during the combined down-regulation Serum levels of FSH, LH, progesterone, testosterone and estradiol are shown in Table 4. The level of LH (P = 0.016) and estradiol (P = 0.001) decreased significantly from Day 1 to Day 70, in spite of reduced levels on Day 1, as 9 women took oral contraceptive pills. Among 11 women initiating treatment in their natural cycle, FSH decreased significantly from 6.5 IU/l (95%CI: 5.6–10.5) on Day 1 to 4.7 IU/l (95%CI: 2.8–5.9) on Day 70 (P = 0.007). The testosterone and progesterone levels did not change significantly during the combined down-regulation. 3.5. Adverse reactions/events No serious adverse reactions or events occurred. Sixteen women (80%) had vaginal spotting/bleeding after the first injection of Zoladex 3.6 mg sc, indicating some degree of flare-up. Two women (10%) had flare-up after the second Zoladex, and one woman (5%) after the third Zoladex, with functional ovarian cysts visible by ultrasound. 4. Discussion To our knowledge this is the first time prolonged combined down-regulation by aromatase inhibitor and GnRH-agonist has been investigated in infertile women with endometriomas/endometriosis undergoing IVF. The volume of the endometriomas and the serum level of CA125 decreased significantly during the treatment indicating inactivation of the disease. Further, it was demonstrated that prolonged combined down-regulation, including daily admin-
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istration of anastrozole, was compatible with IVF pregnancy and the delivery of healthy children following COS. Contradictory results have been published regarding the effect of prolonged GnRH-agonist administration on the size of endometriomas. In some studies the cyst size was not affected [7,8], whereas a reduction after six months of treatment was reported by others [9–11]. Regarding serum CA125, administration of GnRH-agonist for three months significantly reduced levels in patients with moderate and severe endometriosis to 24 U/ml (SD 10) and 26 U/ml (SD 12), respectively [12]. This seems quite similar to the level found after 2.5 months of combined down-regulation in the present study (median 20.5 U/ml, range 10–65). The serum CA125 level is higher during menstruation compared to other parts of the menstrual cycle, especially in women with endometriosis [13]. In the present study, 11 of 20 patients initiated treatment during their menstruation (Day 1), which contributed to elevated CA125 levels measured on Day 1 (Fig. 1). It is therefore important to notice that the CA125 level not only decreased significantly from treatment Day 1 to Day 70, but also from Day 28, where no women had vaginal bleeding, until Day 70. The present study documents the effect of combined aromatase inhibitor and GnRH-agonist to shrink endometriomas and reduce CA125, but an additional effect of adding the aromatase inhibitor, compared to the classical GnRH-agonist alone regiment, remains unproven. Only one randomized controlled trial was published on combined administration of aromatase inhibitor and GnRHagonist [14]. Following surgery for severe endometriosis, six months of anastrozole and goserelin treatment increased the painfree interval and decreased symptom recurrence rates compared to goserelin alone indicating an additional effect of the aromatase inhibitor, but objective parameters, such as the size of endometriomas, has not been recorded in a randomized setting. We therefore still need sufficiently powered randomized trials comparing this new approach to the usual GnRH-agonist treatment. Regarding IVF cycle outcome, a mean fertilization rate of 69.9%, an implantation rate of 14.1%, and a clinical pregnancy rate of 22.7%, were recently reported among 56 women with ultrasounddiagnosed endometriomas >3 cm who followed a standard long agonist protocol [15]. Although several differences exist between this study and the present study, the referred results are remarkable similar. Further, when the IVF/ICSI cycle including prolonged combined down-regulation was compared to a standard IVF/ICSI cycle in 15 patients who served as their own controls, neither the number of oocytes retrieved, the fertilization rate, nor the number of embryos was improved. The present data, therefore, do not prove that prolonged combined aromatase inhibitor and GnRH-agonist down-regulation improves IVF outcome. Interestingly, 9 of the 20 patients (45%) conceived, but 6 of these pregnancies were biochemical pregnancies (n = 4) or early
Table 4 Serum endocrinology during treatment (n = 20). Day 1a
FSH (IU/l) LH (IU/l) Progesterone (nmol/l) Testosterone (nmol/l) Estradiol (nmol/l)
4.5 (0.9–6.5) 2.7 (0.2–5.1) 1.5 (1.0–2.3) 1.1 (0.8–1.7) 0.12 (0.05–0.15)
Day 28
3.7 (2.1–6.3) 0.5 (0.2–0.7) 1.5 (1.1–1.8) 1.3 (0.8–1.5) 0.04 (0.04–0.05)
Day 56
4.9 (3.6–6.2) 0.3 (0.2–0.4) 1.3 (1.1–1.6) 1.0 (0.6–1.3) 0.04 (0.04–0.05)
Day 70
Day hCG
Stim 1
Stim 7
4.8 (3.0–5.7) 0.6 (0.4–0.9) 1.5 (1.1–2.0) 1.1 (0.8–1.4) 0.04 (0.04–0.06)
14.7 (9.8–16.6) 0.4 (0.3–0.6) 1.7 (1.3–2.0) 1.3 (0.8–1.6) 0.49 (0.23–1.13)
15.1 (13.7–20.3) 0.5 (0.3–0.9) 2.2 (1.6–2.9) 1.4 (0.9–1.8) 2.92 (1.61–5.06)
Data are presented as median (95%CI). a On Day 1 (treatment Day 1), nine patients (45%) had taken oral contraceptive pills during at least 14 days to control treatment start. Among the 11 patients that began treatment in their natural menstrual cycle (Days 2–4) the hormonal levels on Day 1 were: FSH 6.5 (5.6–10.5), LH 5.1 (3.2–10.0), progesterone 1.5 (1.0–3.7), testosterone 1.3 (0.7–2.1) and estradiol 0.15 (0.12–0.17).
K. Lossl et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 144 (2009) 48–53
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spontaneous abortions (n = 2), and only 3 patients (15%) reached delivery. This could be accidental, but an impaired quality of the oocytes and/or the endometrium following prolonged combined down-regulation must be considered. Short-term use of aromatase inhibitor before ovarian stimulation increases the androgen level in preovulatory follicular fluid [16,17]. Further, very low serum estradiol and LH levels were noted during the combined downregulation, Table 4. It has earlier been suggested that the estradiol to androgen ratio was higher in follicles containing an oocyte with a better developmental potential [18,19]. The present protocol may therefore benefit from withdrawal of the aromatase inhibitor one week before initiating exogenous gonadotrophin stimulation allowing a ‘‘washout period’’ securing clearance of the aromatase inhibitor with an half-life of 40 h (anastrozole) [20]. Low endogenous LH levels and lack of exogenous LH support have been implicated in less high-quality embryos for cryopreservation [21], lower pregnancy rate [22,23], and an increased miscarriage rate [24]. Limited literature exists on prolonged GnRHagonist down-regulation followed by rFSH stimulation. A high cumulative IVF pregnancy rate after six months of goserelin downregulation and ovarian stimulation with rFSH has been reported [25], but it was not reported in which cycle number the pregnancies occurred leaving it unknown how many occurred directly after the prolonged agonist down-regulation. It might have been beneficial to use hMG or rLH supplementation during the ovarian stimulation in the present study. Several unexpected incidences of flare-up were noted after the second and third administration of goserelin 3.6 mg. The profound suppression of estradiol during the combined treatment may activate pituitary FSH secretion to a higher extent than treatment with the agonist alone. One possibility to overcome this could be reduced intervals between goserelin administrations. Based on the issues discussed above, we propose an experimental protocol in infertile endometriosis patients with shortened intervals between depot GnRH-agonist administrations, and cessation of the aromatase inhibitor one week before COS by hMG. This experimental protocol should be tested in a randomized study design including a control group receiving only the depot GnRH-agonist. In the present study the diagnosis of endometrioma was made by transvaginal ultrasound by two independent physicians. Several investigations have demonstrated the validity of ultrasound to diagnose endometriomas [26,27]. Further, the endometriomas had persisted though at least three months, also in patients with a previous histopathological diagnosis of endometriosis, to reduce the risk of misdiagnosing a haemorrhagic cyst as an endometrioma. In conclusion, 2.5 months of combined aromatase inhibitor and GnRH-agonist administration significantly decreased the volume of endometriomas and the serum level of CA125 in infertile women with endometriomas. IVF following the combined down-regulation was compatible with pregnancy and delivery of healthy children, but a high pregnancy loss was noted. Possible enhanced endometriosis inactivation and signs of improved IVF cycle outcome compared to down-regulation by the agonist alone need to be evaluated in a controlled study design. Conflict of interest None. Acknowledgements The authors thank Dr. J. Ingemanssen and Dr. A. Lindhard for their help in recruiting patients. Merck Serono is thanked for an
unrestricted educational grant supporting Kristine Lossl. AstraZeneca is thanked for covering the costs of Zoladex.
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