The aromatase inhibitor letrozole increases the concentration of intraovarian androgens and improves in vitro fertilization outcome in low responder patients: A pilot study

IN VITRO FERTILIZATION The aromatase inhibitor letrozole increases the concentration of intraovarian androgens and improves in vitro fertilization outcome in low responder patients: a pilot study Juan A. Garcia-Velasco, M.D.,ⴱ Luis Moreno, M.D., Alberto Pacheco, Ph.D., Alfredo Guillén, M.D., Luis Duque, M.D., Antonio Requena, M.D., and Antonio Pellicer, M.D. IVI-Madrid, Rey Juan Carlos University, Madrid, Spain

Objective: To evaluate the impact of aromatase inhibitors as adjuvant treatment in IVF cycles on intraovarian androgens and cycle outcome. Design: Observational, pilot study. Setting: University-affiliated IVF unit. Patient(s): One hundred forty-seven low responder patients with a previous canceled IVF cycle; 71 patients were treated with letrozole 2.5 mg plus a high-dose FSH/hMG-antagonist regimen, and 76 patients were similarly treated but letrozole was not employed. Intervention(s): In vitro fertilization treatment with an antagonist FSH/hMG protocol with or without letrozole was administered during the first 5 days of stimulation; hormones were evaluated in both serum and follicular fluid. Main Outcome Measure(s): Number of oocytes retrieved, fertilization rate, implantation rate, and pregnancy rate; androstenedione, T, E2, and P values in serum and follicular fluid. Result(s): Letrozole-treated patients showed significantly higher levels of follicular fluid T and androstenedione (80.3 vs. 43.8 pg/mL and 57.9 vs. 37.4 mg/mL, respectively). Similarly, these patients had a higher number of oocytes retrieved (6.1 vs. 4.3) and a higher implantation rate (25% vs. 9.4%) despite similar doses of FSH/hMG (3,627 vs. 3,804 IU). Conclusion(s): Adding 2.5 mg of letrozole to a high-dose FSH/hMG antagonist protocol increases intraovarian androstenedione and T concentration and improves IVF cycle outcome in poor responder patients. (Fertil Steril威 2005;84:82–7. ©2005 by American Society for Reproductive Medicine.) Key Words: Poor response, IVF, aromatase inhibitors

Low ovarian response to controlled ovarian hyperstimulation (COH) is still a major concern in assisted reproduction. Poor response to gonadotropins is strongly related to diminished ovarian reserve and advanced maternal age, as both have a direct and profound impact on the success of assisted reproductive technologies. It has been shown that diminished ovarian reserve affects mainly egg production rather than egg quality, a characteristic strongly influenced by maternal age (1, 2). Among the different predictors of poor ovarian response in IVF cycles, age, basal follicle-stimulating hormone (FSH), and antral follicle count seem to perform the best (3, 4). However, some young patients with normal FSH concenReceived September 16, 2004; revised and accepted January 24. 2005. Reprint requests: Juan A Garcia-Velasco, M.D., IVI-Madrid, Santiago de Compostela 88, 28035 Madrid, Spain (FAX: 91-386-7133; E-mail: [email protected]).

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trations present repeated low responses to aggressive stimulation protocols. Obviously, ovarian response to stimulation is the ultimate test (5). In these young patients with either elevated basal FSH or low antral follicle count or a previous cycle with a low response, different strategies have been assayed to improve their outcome. The majority of these strategies aim to recruit a higher number of follicles either by increasing the dose of gonadotropins, by reducing the dose of GnRH analogs, or even by optimizing the endogenous FSH flare effect (6 – 8). Even though it may increase ovarian response, implantation rates remain the same (9, 10). A lower expression of FSH receptor in the granulosa cells from low responder patients has been demonstrated (11). Contrary to this situation are patients with polycystic ovaries (PCOs), who show a hyperexpression of FSH receptor in their follicular granulosa cell population, probably the reason

Fertility and Sterility姞 Vol. 84, No. 1, July 2005 Copyright ©2005 American Society for Reproductive Medicine, Published by Elsevier Inc.

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why they show a tendency to hyperrespond to COH. As these PCO patients show high serum and follicular fluid LH and androgens concentrations (12), we might speculate that inducing a temporary and reversible PCO-like condition in the ovaries of poor responder patients could enhance their follicular recruitment and development. Letrozole is a potent and highly specific nonsteroidal aromatase inhibitor that initially was approved for use in postmenopausal women with breast cancer to suppress estrogen production (13). It inhibits the aromatase enzyme by competitively binding to the heme of the cytochrome P450 subunit of the enzyme resulting in a blockade of androgens conversion into estrogens with a subsequent increase in intraovarian androgens (14). Letrozole, at doses of 1–5 mg/ day, inhibits aromatase activity by 97%–99% (15). A recent study has shown that androgens, in addition to serving as precursors for ovarian estrogen synthesis, also have a fundamental trophic role in primate ovarian follicular development by augmentation of FSH receptor expression on granulosa cells (16). This mechanism may explain the frequent hyperresponse to ovarian stimulation of PCO patients. In fact, previous work has shown that aromatase inhibition improves ovarian response to FSH in poor responder patients undergoing ovulation induction and intrauterine insemination (17). Only limited, retrospective data are available in IVF/intracytoplasmic sperm injection cycles, suggesting that empirically adding an aromatase inhibitor may benefit patients who failed a previous cycle (18, 19). However, no evidence is provided to justify their results. The specific aims of this prospective, pilot study were [1] to assess whether aromatase inhibition with letrozole improves ovarian response and IVF cycle outcome in low responder patients undergoing IVF and [2] to quantify serum and follicular fluid concentration of E2, T, and androstenedione in both groups to evaluate the effect of letrozole. MATERIALS AND METHODS Study Design This study included a total of 147 IVF cycles performed in 147 low responders treated at our institution between November 1, 2002, and February 28, 2004. The study was approved by our institution’s ethics committee, and all couples were required to sign a written informed consent after the provision of complete information. To be included in this study, patients had to have at least one previous canceled IVF attempt in which four or fewer follicles 16 mm in diameter were obtained and/or serum E2 levels were ⱕ500 pg/mL and basal FSH concentrations were ⬍12 IU/mL (8). There were no exclusion criteria and there was no age limit. The canceled cycle was stimulated with a long protocol combined with high doses of FSH/hMG, as described elsewhere (6). Briefly, after pituitary desensitization with a GnRH agonist (triptorelin; Decapeptyl 0.1 mg, Ipsen Pharma, Barcelona, Spain), basal vaginal ultrasound Fertility and Sterility姞

was performed to ascertain ovarian quiescence on the first 3 days of menses. Then ovarian stimulation was started with recombinant FSH (Gonal F, Serono, Madrid, Spain) 225 IU/day together with 150 IU of highly purified hMG (Menopur, Ferring S.A., Madrid, Spain) for the first 3 days, and then individual dose adjustments were done as required, according to serum E2 concentrations and ovarian response (mean gonadotropin dose in the previous cycle, 3,400 ⫾ 181 IU). During their next cycle, the patients were informed about the possibility of being included in the study by adding or not adding letrozole 2.5 mg to the first 5 days of ovarian stimulation. Seventy-one patients were treated in 71 cycles with a high-dose regimen and letrozole, and 76 were treated in 76 cycles with a high-dose regimen alone. Successive ovarian stimulation was separated by a minimum of 2 or more months to avoid any potential source of error, as this is our routine clinical practice. Patients and Stimulation Protocol The etiologies of infertility were 41% male factor, 12% tubal disease, 15% unexplained, and 32% a combination of male and female factors. No patient had any uterine anomaly. The etiology of infertility was equally distributed between groups. All patients had regular menstrual cycles every 26 –32 days and were not taking any medication. The mean (⫾SEM) age of the patients included was 37.1 ⫾ 0.5 years, and the body mass index was 22.9 ⫾ 0.5 kg/m2, with no differences between groups. The mean duration of infertility was 3.5 ⫾ 0.7 years (range, 1– 6 years). Stimulation Protocols The protocol for ovarian stimulation was initiated with the administration of an oral contraceptive pill the month before the cycle (0.05 mg ethinylestradiol ⫹ 25 mg levonorgestrel, Neogynona, Schering AG, Berlin, Germany). Serum E2 concentrations ⬍60 pg/mL (220 pmol/L) and negative findings (absence of ovarian cysts ⬎10 mm diameter) on vaginal ultrasound scans performed on cycle day 1 or 2 were used to define ovarian quiescence. If a cyst ⬎10 mm diameter was observed, then a serum E2 concentration ⬍60 pg/mL was sufficient to confirm ovarian quiescence. If serum E2 concentrations were beyond the cut-off point, the patient was excluded from the study. Briefly, on days 1– 4 of ovarian stimulation, 225 IU of recombinant FSH (rFSH) (Gonal-F 75; Serono) was administered together with 150 IU of highly purified hMG (Menopur, Ferring). Beginning on day 5, rFSH/hMG was administered on an individual basis according to serum E2 concentrations and transvaginal ovarian ultrasound scans. For the first 5 days of stimulation, 71 patients were additionally treated with letrozole (Femara, Novartis, Barcelona, Spain) 2.5 mg/day. Starting when leading follicles reached 14 mm in mean diameter, 0.25 mg of the GnRH antagonist ganirelix (Or83

TABLE 1 Clinical characteristics of patients treated with a high-dose FSH/hMG-antagonist regimen with or without letrozole.

Age (yr) Body mass index (kg/m2) Day 3 FSH (IU/mL) Day 3 LH (IU/mL) Day 3 estradiol (pg/mL) Stimulation days Total FSH ⫹ hMG dose (IU) E2 day of hCG No. of follicles ⱖ 16 mm Endometrial thickness (mm)

Letrozole (n ⴝ 71)

Control (n ⴝ 76)

P

36.5 ⫾ 0.5 22.5 ⫾ 0.4 9.5 ⫾ 0.4 4.5 ⫾ 0.4 60.1 ⫾ 0.1 9.3 ⫾ 0.3 3,627 ⫾ 116 770 ⫾ 67 3.7 ⫾ 0.2 9.6 ⫾ 0.5

37.4 ⫾ 0.4 23.2 ⫾ 0.5 10.1 ⫾ 0.5 5.1 ⫾ 0.3 58.5 ⫾ 5.7 8.9 ⫾ 0.2 3,804 ⫾ 127 813 ⫾ 60 3.2 ⫾ 0.2 9.8 ⫾ 0.3

.16 .25 .32 .18 .84 .24 .31 .63 .16 .69

Garcia-Velasco. Letrozole in poor responder JVF patients. Fertil Steril 2005.

galutran, N.V. Organon, Oss, The Netherlands) was administered on a daily basis to avoid premature ovulation until the day of hCG. Human chorionic gonadotropin (Ovitrelle, Serono) 250 mg was administered when three or more follicles reached 17 mm in mean diameter, and oocyte retrieval was scheduled for 36 hours later. The standard IVF procedure has been described elsewhere (6). All embryos were transferred on day 3 under ultrasound guidance. All patients received P supplementation with 200 mg b.i.d. of vaginal micronized P (Progeffik; Effik Laboratories, Madrid, Spain) as luteal support. A serum ␤-hCG analysis was done 12 days after the ET, and ultrasound scan was scheduled 10 days later to confirm the diagnosis of clinical pregnancy (presence of gestational sac). Sample Collection and Processing The content of all mature follicles (⬎14 mm diameter) containing the cumulus-oocyte complex was collected into sterile plastic tubes by electronic suction. Only follicular fluid from follicles in which an oocyte was clearly identified was used for the study. After isolation of the oocyte, the clear follicular fluid from each patient was pooled and immediately centrifuged (1500 g) for 10 minutes to separate out cellular contents and debris. Follicular fluid supernatant was then transferred to sterile polypropylene tubes and frozen at ⫺20°C until taken for analysis. As a wide interfollicular variation in intrafollicular steroid concentrations has been reported (this reflects interfollicular asynchrony during ovarian stimulation for IVF) (20, 21), the decision was made to use pooled aspirated follicular fluid from each patient in an attempt to assess whole ovarian production, as previously recommended. Hormone Measurements The samples were stored at ⫺20°C in aliquots for subsequent analysis. Serum and follicular fluid E2 and P were 84

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analyzed using a commercially available microparticle enzyme immunoassay kit (Abbot Laboratories, Abbot Park, IL). Inter- and intra-assay coefficients of variation for E2 at a concentration of ⬍40 pg/mL were 2.8% and 4.3%, respectively. The serum P kit had a sensitivity of 0.2 ng/mL, with inter- and intraobserver coefficients of variation of 3.9% and 9.6%, respectively. Serum and follicular fluid T and androstenedione were evaluated by radioimmunoassay (Diagnostic Systems, Webster, TX). Inter- and intra-assay coefficients of variation for T were 8.4% and 7.8%, respectively, and for androstenedione, 6% and 4.3%. Statistical Analysis Data were expressed as mean ⫾ SEM. Student’s t-test and the ␹2 test were used as appropriate. P⬍.05 was considered statistically significant. The statistical analysis was performed with Sigmastat for Windows, version 2.0 (Jandel Scientific, San Rafael, CA). RESULTS Clinical Outcome Patients were comparable in terms of age (37.4 vs. 36.5 years), body mass index (23.2 vs. 22.5 kg/m2), and day 3 serum FSH, LH, and E2 levels (Table 1). No significant differences among groups were found in terms of days of stimulation (8.9 vs. 9.3), mean FSH/hMG dose administered (3,804 vs. 3,627 IU), mean serum E2 levels on the day of hCG (813.5 vs. 770.1 pg/mL), endometrial thickness (9.8 vs. 9.6 mm), and cancellation rate due to low response (19.7% vs. 15.5%). Despite there being no difference in the number of mature follicles observed (3.3 vs. 3.7), the number of oocytes retrieved was significantly higher in the study group (4.3 vs. 6.1; P ⫽ .033), with a similar fertilization rate between groups (62% vs. 57.7%) (Table 2). Moreover, we found a marked difference in the implantation rate being signifi-

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TABLE 2 Cycle outcome characteristics in patients treated with a high-dose FSH/hMG-antagonist regimen with or without letrozole. Letrozole (n ⴝ 71)

Control (n ⴝ 76)

P

6.1 ⫾ 0.4 68.2 2 ⫾ 0.1 22.4 41.7 25 15.5 43.7 20 46.7

4.3 ⫾ 0.3 63.3 2.3 ⫾ 0.1 15.2 28.9 9.4 19.7 44.7 7.7 7.7

.03 .51 .09 .39 .36 .009 .82 .97 .6 .04

No. of oocytes retrieved Fertilization rate (%) No. of embryos transferred PR/cycle PR/transfer Implantation rate Cycle cancellation due to low response Total cycle cancellation Miscarriage rate Multiple pregnancy rate (twins) Garcia-Velasco. Letrozole in poor responder JVF patients. Fertil Steril 2005.

cantly higher in the letrozole group (9.4% vs. 25%; P ⫽ .009). The pregnancy rate per transfer also was higher in the letrozole group (41.6%) when compared with the control group (28.9%), but this finding was not significant, although the multiple pregnancy rate was statistically significantly higher in letrozole-treated patients (46.7% vs. 7.7%; P ⫽ .04).

had been treated with letrozole (80.3 vs. 43.8 pg/mL and 57.9 vs. 37.4 mg/mL, respectively; P ⫽ .004). Although a trend was observed toward higher serum levels of T and androstenedione in patients who had received letrozole (1.7 vs. 1.1 pg/mL and 1.9 vs. 1.4 ng/mL, respectively), differences were not significant.

The miscarriage rate was comparable between groups (20% vs. 8%, P ⫽ NS). The global cancellation rate in these poor prognosis patients, including low response, failed fertilization, embryo blockage, or no embryos available for transfer, was similar in both groups of patients (43.7% vs. 44.7%; P ⫽ NS) (Table 2).

DISCUSSION Low response to COH is the clinical expression of diminished ovarian reserve, and it represents one of the biggest challenges to the clinician. It has been shown that patients with a poor ovarian response in the first IVF cycle do not necessarily exhibit a low response in subsequent cycles, especially patients with unexpected poor response (22). This subset of patients, young unexpected low responders with normal day 3 FSH values, might be the best to benefit from new strategies that make accessible the follicles still available.

Hormonal Determinations Serum and/or follicular fluid E2 and P levels were comparable in patients that had or had not been treated with letrozole (Table 3). Interestingly, both follicular fluid T and androstenedione were significantly higher in patients that

There is a lack of uniform definition of a poor responder, so comparing treatments becomes a difficult task. Even

TABLE 3 Serum and follicular fluid hormonal values obtained in patients treated with and without letrozole. Letrozole (n ⴝ 20)

Control (n ⴝ 21)

P

384 ⫾ 54 3,884 ⫾ 586 2.8 ⫾ 0.5 12.1 ⫾ 1.1 1.7 ⫾ 0.3 80.3 ⫾ 9.6 1.9 ⫾ 0.5 57.9 ⫾ 6.1

485 ⫾ 45 4,145 ⫾ 568 3.7 ⫾ 0.4 12.5 ⫾ 0.8 1.1 ⫾ 0.1 43.8 ⫾ 5.9 1.4 ⫾ 0.1 37.4 ⫾ 2.4

.16 .75 .15 .08 .07 .004 .29 .004

Serum E2 (pg/mL) Follicular fluid E2 (pg/mL) Serum P (ng/mL) Follicular fluid P (ng/mL) Serum T (pg/mL) Follicular fluid T (pg/mL) Serum androstenedione (ng/mL) Follicular fluid androstenedione (ng/mL) Garcia-Velasco. Letrozole in poor responder JVF patients. Fertil Steril 2005.

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more, some patients may respond poorly in a first cycle, but not necessarily in a second cycle because of random fluctuations, a phenomenon known as regression to the mean normal response (23). In our practice, we have observed that retrieving fewer than five mature oocytes significantly affects IVF cycle outcome (unpublished observation), so we considered patients with a previous cycle canceled because of low response according to these criteria eligible for this new protocol. We did not attempt to determine outcome in relation to screening test (i.e., basal FSH), but rather used the pragmatic approach of a previous canceled cycle due to low response, which has been shown to be a sensitive as well as dynamic test for ovarian reserve (24). This simple approach obtained not only better results in terms of implantation rate, but also an increase in intraovarian androgen levels that may justify the outcome. Considering that increasing intrafollicular androgen concentrations may induce a temporary reversible PCO-like condition, as PCO women usually show a hyperresponse to FSH/hMG and their granulosa cells are hyperresponsive and develop LH receptors earlier than non-PCO women (5, 12), we evaluated in this pilot study the effect of this strategy in poor responder patients by adding letrozole for the first 5 days of ovarian stimulation in IVF patients. We observed higher levels of follicular fluid, T, and androstenedione in patients who exhibited a significantly higher number of oocytes retrieved as well as a higher implantation rate, hypothesizing that letrozole may improve the prognosis of these patients. Considering that letrozole was administered in cycle days 1–5 and steroids were measured on cycle day 10, it may be acting through FSH/LH receptors indirectly by increasing androgen levels. Intraovarian androgens may have a profound effect on early follicle growth. It has been shown in cultured mouse preantral follicles that androgen treatment stimulates follicle growth (25). In a model closer to humans, intact monkeys treated with androgens increase the number of preantral and small antral follicles (26, 27) as androgens stimulate theca and granulosa cell proliferation and inhibit apoptosis. It seems that this effect is mediated via androgen receptor. In fact, the average primate androgen receptor level in granulosa cells from immature follicles is 4.2-fold higher than in preovulatory follicles (28). Even more, Weil et al. (27) demonstrated a higher expression of androgen receptor gene expression in preantral and antral follicles. The fact that androgen treatment may promote gonadotropin responsiveness in granulosa cells was first introduced by Hillier (29). More recently, Weil et al. (16) described T as stimulating FSH receptor expression by in situ hybridization in primates, suggesting that androgen treatment may promote follicular growth and estrogens biosynthesis. Androgens also stimulate IGF-I and IGF-I receptor gene expression (30), which is known to promote follicular steroidogenesis (31). 86

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There is considerable evidence in the literature to support the close relationship between high levels of circulating androgens and small follicle numbers on ultrasound. Ovarian tissue transplanted into male mice induced larger follicles than the same tissue transplanted into female mice (32). Similarly, when female-to-male transsexuals are treated with androgens, an increased number of nonovulatory antral follicles are seen, similar to those observed in women with PCO syndrome (33). Many of these follicles show healthy steroidogenic and growth characteristics (34, 35). Jonard et al. (36) showed a positive correlation between serum T and androstenedione levels in PCO patients and the number of 2–5 mm follicles. All these data support the trophic effects of androgens in small follicles. If androgens may have a role in developing preantral and antral follicles then directly treating the ovary with androgens could be an attractive hypothesis to improve ovarian response in poor responders. Androgens serve as a substrate for E2 production, promote the growth of small follicles and the proliferation of granulosa and theca cells, and stimulate FSH receptor gene expression as well as IGF-I and IGF-I receptor. However, when concentrations are too high, they can reduce follicular health (37). A recent report has tested the hypothetical benefit of adding transdermal T 15 days before FSH treatment in low responder IVF patients (38). The investigators found a significantly higher number of oocytes in the T group, suggesting a synergistical role of androgens with FSH to promote early follicular recruitment. Using aromatase inhibitors in a conceiving cycle raises some safety issues regarding teratogenicity. We have used letrozole for short-term administration only during the first 5 days of ovarian stimulation after a menstrual period, ruling out the possibility of pregnancy. Also, the short half-life— approximately 2 days— of the drug assures that all of the drug will be completely cleared before the organogenesis period starts (15), in case the ET is successful. In this prospective pilot study, we have demonstrated that aromatase inhibition with letrozole at the beginning of ovarian stimulation significantly increases intraovarian androgen concentrations, a finding that may justify the improved ovarian response. Further randomized, controlled trials should validate these findings. REFERENCES 1. Van Rooij IA, Bancsi LF, Broekmans FJ, Looman CW, Habbema JD, te Velde ER. Women older than 40 years of age and those with elevated follicle-stimulating hormone levels differ in poor response rate and embryo quality in in vitro fertilization. Fertil Steril 2003;79:482– 8. 2. Toner JP. Age⫽egg quality, FSH level⫽egg quantity. Fertil Steril 2003;79:491. 3. Bancsi LF, Broekmans FJ, Eijkemans MJ, de Jong FH, Habbema JD, te Velde ER. Predictors of poor response in in vitro fertilization: a prospective study comparing basal markers of ovarian reserve. Fertil Steril 2002;77:328 –36. 4. Chuang CC, Chen CD, Chao KH, Chen SU, Ho HN, Yang YS. Age is a better predictor of pregnancy potential than basal follicle-stimulating

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22. Klinkert E, Broekmans FJ, Looman CW, te Velde ER. A poor response in the first in vitro fertilization cycle is not necessarily related to a poor prognosis in subsequent cycles. Fertil Steril 2004;81:1247–53. 23. Pantos C, Thornton SJ, Speirs AL, Johnston I. Increasing the human menopausal gonadotropin dose— does the response really improve? Fertil Steril 1990;53:436 –9. 24. Beckers NG, Macklon NS, Eijkemans MJ, Fauser BC. Women with regular menstrual cycles and a poor response to ovarian hyperstimulation for in vitro fertilization exhibit follicular phase characteristics suggestive of ovarian aging. Fertil Steril 2002;78:291–7. 25. Murray AA, Gosden RG, Allison V, Spears N. Effect of androgens on the development of mouse follicles growing in vitro. J Reprod Fertil 1998;113:27–33. 26. Vendola KA, Zhou J, Adesanya OO, Weil SJ, Bondy CA. Androgens stimulate early stages of follicular growth in the primate ovary. J Clin Invest 1998;101:2622–9. 27. Weil SJ, Vendola KA, Zhou J, Adesanya OO, Wang J, Okafor J, Bondy CA. Androgen receptor gene expression in the primate ovary: cellular localization, regulation, and functional correlations. J Clin Endocrinol Metab 1998;83:2479 – 85. 28. Hillier SG, Tetsuka M, Fraser HM. Location and developmental regulation of androgen receptor in primate ovary. Hum Reprod 1997;12: 107–11. 29. Hillier SG. Hormonal control of folliculogenesis and luteinization. In: Findlay JK, ed. Molecular biology of female reproductive system. San Diego: Academic Press, 1994:1–37. 30. Vendola KA, Zhou J, Wang J, Bondy CA. Androgens promote the IGF-I and IGF-I receptor gene expression in the primate ovary. Hum Reprod 1999;14:2328 –32. 31. Demeestere I, Gervy C, Centner J, Devreker F, Englert Y, Delbaere A. Effect of insulin-like growth factor-I during preantral follicular culture on steroidogenesis, in vitro oocyte maturation, and embryo development in mice. Biol Reprod 2004;70:1664 –9. 32. Weissman A, Gotlieb L, Colgan T, Jurisicova A, Greenblatt EM, Casper RF. Preliminary experience with subcutaneous human ovarian cortex transplantation in the NOD-SCID mouse. Biol Reprod 1999;60: 1462–7. 33. Pache TD, Fauser BC. Polycystic ovaries in female-to-male transsexuals. Clin Endocrinol 1993;39:702–3. 34. Pache TD, Hop WC, de Jong JH, Leeventveld RA, van Geldorp H, Van de Kamp TM, et al. 17b-oestradiol, androstenedione and inhibin levels in fluid from individual follicles of normal and polycystic ovaries, and in ovaries from androgen treated female to male transsexuals. Clin Endocrinol 1992;36:565–71. 35. Takayama K, Fukaya T, Sasano H, Funayama Y, Suzuki T, Takaya R, et al. Immunohistochemical study of steroidogenesis and cell proliferation in polycystic ovarian syndrome. Human Reprod 1996;11:1387– 92. 36. Jonard S, Robert Y, Cortet-Rudelli C, Pigny P, Decanter C, Dewailly D. Ultrasound examination of polycystic ovaries: is it worth counting the follicles? Hum Reprod 2003;18:598 – 603. 37. Jonard S, Dewailly D. The follicular excess in polycystic ovaries, due to intraovarian hyperandrogenism, may be the main culprit for the follicular arrest. Hum Reprod Update 2004;10:107–17. 38. Massin N, Cedrin-Durnerin I, Galey-Fontaine J, Coussieu C, BryGauillard H, Hughes JN. Is androgen application beneficial for low responders? Hum Reprod 2004;19(Suppl):20 –1.

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