Hum. Reprod. Advance Access originally published online on March 3, 2006
Human Reproduction 2006 21(7):1884-1893; doi:10.1093/humrep/del052
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Pretreatment with transdermal testosterone may improve ovarian response to gonadotrophins in poor-responder IVF patients with normal basal concentrations of FSH
1 Institut Clínic of Gynecology, Obstetrics and Neonatology and 2 Hormonal Laboratory, Hospital Clínic-Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain
3 To whom correspondence should be addressed at: Institut Clínic of Gynecology, Obstetrics and Neonatology, Hospital Clínic-Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), C/Casanova 143, 08036-Barcelona, Spain. E-mail: jbalasch{at}ub.edu
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Abstract |
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BACKGROUND: Treatment of poor-responder patients to controlled ovarian stimulation for assisted reproduction, who have normal basal FSH concentrations, is one of the most difficult challenges in reproductive medicine. This study investigated the usefulness of testosterone pretreatment in such patients. METHODS: Prospective, therapeutic, self-controlled clinical trial including 25 consecutive infertile patients who had a background of the first and second IVF treatment cycle cancellations due to poor follicular response, in spite of vigorous gonadotrophin ovarian stimulation and having normal basal FSH levels. In the third IVF attempt, all patients received transdermal testosterone treatment (20 µg/kg per day) during the 5 days preceding gonadotrophin treatment. RESULTS: Twenty patients (80%) showed an increase of over fivefold in the number of recruited follicles, produced 5.8 ± 0.4 (mean ± SEM) oocytes, received two or three embryos and achieved a clinical pregnancy rate of 30% per oocyte retrieval. There were 20% cancelled cycles. CONCLUSION: Pretreatment with transdermal testosterone may be a useful approach for women known to be low responders on the basis of a poor response to controlled ovarian stimulation but having normal basal FSH concentrations.
Key words: androgens/IVF/low responders
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Introduction |
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Assisted reproductive technologies (ARTs) are being increasingly used in infertility treatment. ARTs imply the pharmacological induction of multiple follicular recruitment to obtain multiple oocytes and embryos. The most widely used protocol for ovarian stimulation in ART cycles has involved the administration of gonadotrophins under pituitary suppression with GnRH agonists (the so-called ‘long down-regulation protocol’), which not only increases pregnancy and live-birth rates but also allows flexible timing for oocyte recovery and greatly simplifies IVF treatment (Daya, 1997
, 2000; Barbieri and Hornstein, 1999). However, many women are found to respond poorly or not at all to this standard treatment. Such patients are referred to as ‘low or poor responders’, and the incidence of low responders is estimated to be 10–25% of the ART population (Keay et al., 1997; Karande and Gleicher, 1999; Fasouliotis et al., 2000; Tarlatzis et al., 2003). Cycle cancellation seems the most logical criterion to define the poor-responder patient, but even in cases that reach oocyte retrieval the prognosis is poor (Keay et al., 1997; Surrey and Schoolcraft, 2000; Mahutte and Arici, 2002; Tarlatzis et al., 2003).
Low response to ovarian stimulation frequently reflects an age-related decline in reproductive performance (older patients with an abnormal endocrinological profile), but the same phenomenon may occur in young patients (Fasouliotis et al., 2000; Tarlatzis et al., 2003). Some of the latter group of women have so-called ‘occult ovarian failure’ evidenced by elevated FSH serum concentrations (Cameron et al., 1988), but others have normal serum FSH and no apparent reason for repeated low responses to aggressive stimulation protocols (Fasouliotis et al., 2000; Tarlatzis et al., 2003). Since the evolution of ART, the management of these younger women with normal hormonal profile has been one of the most difficult challenges and disappointing issues in reproductive medicine. In fact, it has been stressed that irrespective of the protocol used, the treatment of poor responders (including the idiopathic group with no defined risks) results in a low pregnancy rate, unless the couple makes the difficult decision to use donor oocytes (Keay et al., 1997; Surrey and Schoolcraft, 2000; Mahutte and Arici, 2002; Tarlatzis et al., 2003).
Interestingly, early studies showed that granulosa cell stimulation by FSH is an androgen-modulated process in vitro and recent work in the primate ovary strongly suggests that androgens may influence the responsiveness of ovaries to gonadotrophins (Hillier and De Zwart, 1981; Harlow et al., 1986). In a series of primate studies, findings suggested that androgens appear to be positive regulators of follicular development, as treatment of rhesus monkeys with dihydrotestosterone (DHT) or testosterone augments follicular FSH-receptor expression in granulosa cells (Weil et al., 1998, 1999), promotes initiation of primordial follicle growth (Vendola et al., 1999) and increases the number of growing preantral and small antral follicles (Vendola et al., 1998). Altogether, these studies suggest that androgen treatment may amplify the FSH effects on the ovary. In the clinical setting, it was reported that dehydroepiandrosterone (Casson et al., 2000) or letrozole (an aromatase inhibitor which induces a temporary accumulation of intraovarian androgens) (Mitwally and Casper, 2002; García-Velasco et al., 2005) supplementation improved the estradiol (E2) and/or follicular response to gonadotrophin treatment. Also, two recent studies have reported a positive correlation of serum testosterone levels with some, but not all, parameters of ovarian stimulation in IVF cycles (Frattarelli and Peterson, 2004; Barbieri et al., 2005).
On the above evidence, this prospective study was undertaken to investigate the usefulness of testosterone pretreatment in women with normal FSH serum concentrations who had two consecutive cancelled IVF cycles because of insufficient ovarian response in spite of vigorous gonadotrophin ovarian stimulation.
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Materials and methods |
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Patients studied
This was a prospective, therapeutic, self-controlled clinical trial including 25 women undergoing IVF treatment at the Fertility Unit of the Hospital Clínic of Barcelona. All patients gave informed consent to participate in the study, which was approved by the ethics committee of our hospital. They were consecutive women who had a background of the first and second IVF treatment cycles cancelled because of poor follicular response in spite of having normal basal FSH concentrations and fulfilling the inclusion criteria reported below. In their third IVF attempt, all patients received transdermal application of testosterone preceding gonadotrophin ovarian stimulation. Thus, each patient served as a control for herself and three consecutive IVF cycles were compared in every case. The period between the first and third IVF cycles was always within 13 months.
All the subjects were regularly menstruating (menstrual cycles of 26–32 days), premenopausal women aged 31–39 years (mean ± SEM, 35.6 ± 0.4 years) at the time of their third IVF attempt and having a normal BMI of 21.2–27.4 kg/m2 (mean ± SEM, 24.7 ± 0.2 kg/m2). All the women had both ovaries, no previous ovarian surgery and normal ovulatory function according to midluteal serum progesterone concentrations and regular menses. None of them had occult ovarian failure on the basis of their basal FSH concentration of <10 IU/l [Standard International Reference Preparation (IRP) 78/549] measured in the cycle preceding IVF. No patient had received any hormone therapy between the first and third IVF attempts. Patient indications for IVF/intracytoplasmic sperm injection included the following main diagnosis: male factor infertility (14 patients, 56%), unexplained infertility (5 patients, 20%), tubal infertility (4 patients, 16%) and mild endometriosis (2 patients, 8%).
Stimulation regimens
In the first IVF treatment cycle, ovarian stimulation was carried out with recombinant human FSH (r-hFSH) under pituitary suppression with GnRH agonist according to a routinely used protocol previously reported (Balasch et al., 2001). Pituitary suppression was achieved by s.c. administration of leuprolide acetate (Procrin; Abbott Laboratories, Madrid, Spain). This treatment was started in the midluteal phase of the previous cycle and given 1 mg daily, then reduced to 0.5 mg after ovarian arrest was confirmed and continued until the administration of HCG. Gonadotrophin stimulation of the ovaries was started when serum E2 concentrations declined to <50 pg/ml and a vaginal ultrasonic scan showed an absence of follicles >10 mm diameter. On days 1 and 2 of ovarian stimulation, 450 and 300 IU per day of r-hFSH (Gonal-F, Serono S.A., Madrid, Spain), respectively, were administered s.c. On days 3 and 4 of ovarian stimulation, 150 IU per day of r-hFSH were administered to each patient. From day 5 onwards, r-hFSH was administered on an individual basis according to the ovarian response as assessed by sequential transvaginal ultrasonography and serum E2 measurements.
In the second IVF treatment cycle, high-dose gonadotrophin in association with a reduced dose leuprolide protocol was used for ovarian stimulation. Patients received 0.5 mg/day of leuprolide from the midluteal phase until pituitary suppression was achieved and then 0.25 mg/day thereafter. On days 1 and 2 of ovarian stimulation, 300 IU per day of r-hFSH (Gonal-F, Serono S.A.) were administered together with four ampoules of HMG (75 IU FSH and 75 IU LH per ampoule; Pergonal, Serono S.A.) given i.m. On days 3 and 4 of ovarian stimulation, four ampoules per day of HMG were administered to each patient. From day 5 onwards, HMG was administered on an individual basis according to the ovarian response.
In the third IVF attempt, the patients received exactly the same standard long down-regulation protocol (including gonadotrophin type and dose on days 1–4 of ovarian stimulation) used in their first IVF treatment cycle but now including testosterone therapy during the 5 days preceding gonadotrophin treatment. Testosterone therapy was commenced the day when pituitary–ovarian suppression was evidenced (Figure 1). Transdermal testosterone treatment was carried out using a daily single patch with a 2.5 mg/day nominal delivery rate of testosterone (Androderm 2.5 mg, Cepa Schwarz Pharma, Madrid, Spain) which was applied on the thigh at night and removed always at 09.00 hours in the morning. This transdermal delivery system maintains stable testosterone levels within narrow ranges with little within- and between-subject variation, provides a highly controllable way of delivering testosterone reliably, and the hormonal dose administered can be modified according to the duration of patch application (Buckler et al., 1998; De Sanctis et al., 1998; Mazer, 2000). On the basis of previous experimental studies in primates (Vendola et al., 1998, 1999), we chose to use testosterone 20 µg/kg per day for 5 days. Thus, in each patient, the patch was applied at night at a time aimed to leave it in place for a predetermined number of hours to provide the desired daily dose of testosterone [e.g. in a woman weighing 60 kg and needing 1200 µg/day, the patch was used for 12 h (0.1 mg/h delivery rate x 12 h = 1.2 mg or 1200 µg) and thus applied at 21.00 hours].
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Gonadotrophin ovarian stimulation was started the day following last testosterone patch application. The criteria for HCG administration (5000 IU; Profasi, Serono S.A.) were the presence of two or more follicles
18 mm in diameter with 4 follicles measuring 14 mm in association with a consistent rise in serum E2 concentration. According to previously reported criteria (Peñarrubia et al., 2005a,b), the cycle was cancelled when there were <3 follicles with diameter 14 mm after 8–9 days of gonadotrophin therapy (early cancellation) or after 4–5 additional treatment days without attaining, or the imminent prospect of attaining, the criteria for HCG administration (late cancellation).
Oocyte aspiration was performed with vaginal ultrasonography 35–36 h after HCG administration. Embryo grading was recorded according to published criteria (Veeck, 1999); embryos of grades 1 or 2 were considered high quality. Two to three days after oocyte recovery, up to three embryos per patient (depending on the age of the patient, the indication for IVF and the number and quality of embryos available per replacement) were replaced. Two additional doses of 2500 IU of HCG on the days of embryo transfer and 3 days later were given to supplement the luteal phase. Pregnancy was diagnosed by increasing serum concentrations of 
-HCG after embryo transfer and the subsequent demonstration of an intrauterine gestational sac by ultrasonography.
Monitoring, hormone analyses and ultrasonography
In all patients, blood samples for hormone measurements were obtained at seven sequential study points during the testosterone supplemented IVF cycle (Figure 1): the early follicular phase (cycle day 3) of the spontaneous menstrual cycle preceding IVF (study point 1); the day when pituitary suppression was shown (study point 2); on day 2 of testosterone treatment (study point 3); the day following last testosterone patch application (study point 4); on the fifth day of gonadotrophin therapy (study point 5); on the seventh day of gonadotrophin treatment (study point 6) and on day 9–10 of ovarian stimulation with gonadotrophins (study point 7). Study point 1 was considered as baseline. Each day one blood sample was drawn between 09.30 and 10.00 hours, and for this study two serum aliquots were obtained. Baseline FSH, LH and E2, as well as E2 serum concentrations during ovarian stimulation, were measured daily in one of the serum aliquots for clinical monitoring, and the second aliquot was stored at –20°C for later measurements of testosterone, LH, androstenedione, insulin-like growth factor-I (IGF-I) and sex hormone-binding globulin (SHBG). Frozen blood samples from each patient were examined in one run.
Hormones were measured using commercially available kits. FSH and LH serum concentrations were measured by an immunoenzymatic assay with two monoclonal antibodies (Immuno 1, Technicon; Bayer, Tarrytown, NY, USA). Data are expressed in terms of IRP 78/549 and 68/40 for FSH and LH respectively. The sensitivity of the assays was 0.1 IU/l for FSH and 0.3 IU/l for LH, and the inter-assay coefficients of variation (CV) were 2.7 and 3.1%, respectively. E2 and testosterone concentrations in serum were estimated by a competitive immunoenzymatic assay (Immuno 1, Technicon; Bayer). The sensitivity was 10 pg/ml for E2 and 8 ng/dl for testosterone, and the inter-assay CV were 5 and 9.5%, respectively. Androstenedione in serum was measured by a competitive radioimmunoassay (Diagnostic Systems Laboratories, Webster, TX, USA); the sensitivity of the assay was 10 ng/dl and the inter-assay CV 12%. Serum total IGF-I concentrations were measured by an immunoradiometric assay with two monoclonal antibodies, after acid-ethanol extraction (Immunotech, Beckman Coulter Company, Marseille, France). The standard was calibrated against the WHO IRP 87/518. The sensitivity was 30 ng/ml and the intra- and inter-assay CV were 5.7 and 8.6%, respectively, for values of 360 ng/ml. Serum levels of SHBG were determined using an electrochemiluminescence immunoassay with two monoclonal antibodies (Roche Elecsys, Mannheim, Germany). The lower limit of detection was 0.350 nmol/l and the inter-assay CV was <5%. Total -HCG was measured by a solid-phase two-site chemiluminiscent enzyme immunometric assay standardized against the Third International Standard 75/537 (Immulite; Diagnostic Products, Los Angeles, CA, USA) with a detection limit of 2 IU/l. The inter-assay CV was 5.8%.
All ultrasonic scans were performed by a single investigator (J.P.) using a Toshiba Eccocee SAA-340 A/EF unit (Toshiba, Tokyo, Japan) equipped with a 5–7 MHz endovaginal probe (PVF-641VT). The ovary was examined by scanning from the outer to the inner margin, as described previously (Pache et al., 1990; Scheffer et al., 1999). Round or oval echo-free structures in the ovaries were regarded as follicles and were counted and measured as such. Antral follicle count was performed at study points 1, 2, 3 and 4 during the testosterone supplemented IVF cycles. For the antral follicle count, the numbers of follicles in both ovaries were added and in the statistical analysis, follicles with a diameter of up to 10 mm were included. This is based on the finding that follicle size can vary up to 10 mm before the dominant follicle is identified and, by including follicles up to 10 mm, the antral follicle cohort is determined at its maximal size (Pache et al., 1990; Scheffer et al., 1999). The limit of sensitivity was 2 mm.
Statistics
Data were analysed by Statistics Package for Social Sciences (SPSS) statistical software using the Mann–Whitney U-test and Friedman test as appropriate. Serum hormone concentrations were calculated in each cycle as the area under the curve (AUC), each curve being delineated by the seven points mentioned above. The AUC, which was calculated according to the trapezoidal rule, corresponds to an integrated hormone concentration for the chosen days. Integrated concentrations were compared between treatment groups by the Mann–Whitney U-test. Results are expressed as mean ( SEM. P < 0.05 was considered significant.
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Results |
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Results are presented in Table I–
IV and Figure 2. Table I shows basal hormone serum concentrations, gonadotrophin treatment and ovarian response in the three consecutive IVF treatment cycles in the 25 patients studied. Basal hormone measurements as well as days to ovarian arrest were similar in the three cycles studied. E2 peak serum concentrations and the number of follicles recruited were significantly higher in treatment cycle 3 than in the two previous IVF treatment cycles. The amount of gonadotrophin used was significantly lower in cycles 1 and 3 than in cycle 2. The duration of gonadotrophin therapy tended to be shorter in the third treatment cycle but differences did not reach statistical significance.
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Oocyte retrieval and IVF outcome in the testosterone supplemented cycle in the 25 patients are summarized in Table II. There were five early cancelled cases (20%) and 20 patients (80%) who received HCG and underwent oocyte retrieval. There was an average of 8.5 ± 0.7 follicles punctured. Retrieved oocytes averaged 5.8 ± 0.4, with 4.6 ± 0.4 mature oocytes per punctured cycle. The mean number of embryos available per patient was 3.5 ± 0.3. Two to three embryos were replaced into the uterus in the 20 women and all of them received at least one high quality embryo. There were six clinical pregnancies including three pairs of twins, giving a 24% pregnancy rate per started cycle, a 30% pregnancy rate per oocyte retrieval and per embryo transfer and a 16.6% implantation rate. One single pregnancy ended in a first trimester spontaneous abortion and the remaining five patients had successful live births. No patient developed ovarian hyperstimulation syndrome. No local or systemic side effects related to the use of transdermal testosterone patches were reported.
Table III shows patient characteristics, gonadotrophin treatment and ovarian response in cancelled and non-cancelled IVF cycles where testosterone supplementation was used. Mean age, mean BMI and basal FSH, LH and E2 serum concentrations were similar in both group of patients. The antral follicle count at baseline (study point 1) and study point 2 was almost identical in the two groups of patients but the antral follicle number during testosterone treatment showed a higher increase in non-cancelled cycles, the difference almost reaching statistical significance (P = 0.06) at study point 4. There were no differences regarding time for ovarian arrest and amount and duration of gonadotrophin stimulation between cancelled and non-cancelled cycles but, as expected, E2 peak concentrations and the number of follicles recruited were significantly higher in the non-cancelled group (P < 0.001 for both).
Figure 2 shows sequential serum hormone measurements in the testosterone supplemented cancelled and non-cancelled IVF cycles. Baseline hormone concentrations were similar in both study cycles and within the normal range in all patients included. Hormone concentrations calculated as the AUC are presented in Table IV. Serum concentrations of testosterone and LH did not differ between the two groups of patients. There was a marked overlap in testosterone serum levels throughout the study period in cancelled and non-cancelled cycles, and in both groups of patients testosterone serum concentrations were significantly increased at study points 3 and 4 with respect to baseline (study point 1; P < 0.001 for both groups). However, LH serum levels were somewhat higher in the cancelled group. Serum levels of SHBG throughout the period of study were similar in cancelled and non-cancelled cycles.
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Discussion |
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The concept that androgens are atretogenic was derived from murine ovary studies and this has been challenged by recent work in primates showing that androgens may have, in fact, some synergistic effects with FSH on follicular growth and development (Vendola et al., 1998
, 1999; Weil et al., 1998, 1999). In this self-controlled clinical trial, we demonstrate a potential benefit of testosterone treatment in improving ovarian response to FSH stimulation in previous poor responders to vigorous gonadotrophin treatment for IVF. The following facts support this contention. First, as much as 80% of patients underwent oocyte retrieval and embryo transfer in the testosterone supplemented IVF cycle. Second, the total amount of gonadotrophin needed to achieve such improved response with testosterone was significantly lower in the IVF cycle 3 as compared with cycle 2 where the dose of gonadotrophin administered was increased with the expectation that these patients would respond to the higher serum levels of gonadotrophins with an increase in the number of follicles recruited. Third, the antral follicle count increased during testosterone treatment, and the number of follicles available for recruitment and development at the time of starting FSH therapy was higher in non-cancelled versus cancelled cycles in the third IVF treatment cycle. Finally, androgen treatment promoted FSH action in those recruitable follicles as suggested by increased serum levels of androstenedione and IGF-I (both considered as markers of ovarian responsiveness to gonadotrophin stimulation) in non-cancelled versus cancelled testosterone supplemented IVF cycles. LH serum levels were somewhat lower in the non-cancelled group of patients having multiple growing follicles during ovarian stimulation than in cancelled cycles due to a poor follicular response. This can be explained on the basis that during ovulation induction, gonadotrophin-stimulated estrogens and inhibins feed back on the hypothalamic-pituitary axis and reduce endogenous gonadotrophin secretion and, thus, the feed back effect is more evident when multiple follicular development occurs.
The current study has several potential limitations but also some strengths. Thus, the self-controlled study design may appear suboptimal because in such a study part of the observed treatment effect may be due to the phenomenon of ‘regression towards the mean’. That is, when an extreme value is remeasured, it tends to be closer to the mean of the original population from which the study subjects were drawn, because the original value was likely to have been unduly influenced by random variation. In other words, if patients were enrolled in a self-controlled study of some new approach when standard treatment appear to be losing efficacy (i.e. when the patients’ conditions are worse than average), some general improvement may occur that has nothing to do with improved therapy (Rose and Barker, 1978; Louis et al., 1986). In ART cycles, the ‘regression towards the mean’ occurs because the low response group might have included women who usually have a normal response but by chance were low in the first cycle. Regression to the mean has been mentioned previously as an explanation for the spurious effect of increasing the dose after a poor response in the first cycle (Pantos et al., 1990; Klinkert et al., 2004). However, the following facts should be considered in this respect.
The self-controlled study design was dictated by selection of a specific group of very difficult responder patients in whom both a well-established and routinely used long down-regulation protocol and a widely used approach for treatment of previous poor responders to the standard ovarian stimulation regimen were applied without any success. In the first ART cycle, patients were treated with a step-down regimen of r-hFSH administration under pituitary suppression. In this tapering regimen the highest dose of FSH is given on stimulation days 1 and 2 (six and four 75 IU FSH ampoules) and is then reduced to two ampoules daily once follicular recruitment has been achieved. This regimen has proved to be clinically efficacious (Davis and Rosenwaks, 1996; Balasch et al., 2001; Peñarrubia et al., 2003) and is further supported by the following. First, it has been shown that for successful induction of multiple folliculogenesis in normally ovulating women, there is a critical period during the early follicular phase of the cycle when FSH values should remain above the physiological level to maximally stimulate follicle recruitment in the primary cohort (Messinis and Templeton, 1990; Lolis et al., 1995). Second, follicles recruited by exogenous FSH require an FSH threshold concentration that is higher than that in the natural cycle (Lolis et al., 1995). Third, marked patient variability exists in FSH thresholds as well as in FSH metabolic clearance and ovarian sensitivity to FSH (Porchet et al., 1994; Ben-Rafael et al., 1995; van Santbrink et al., 1995). Remarkably, in clinical studies, such a threshold level was reached with a single injection of six ampoules of FSH on cycle day 2 and further growth of the follicles was obtained with extra FSH from cycle day 4 onwards at the daily dose of two ampoules (Lolis et al., 1995). The above notwithstanding, all patients failed to respond in their first treatment cycle. In their second attempt of ovarian stimulation for ART, patients were treated with high dose gonadotrophin (including HMG) and a 50% reduction in the standard dose of leuprolide acetate which are both measures proposed for treatment of low responder patients in an attempt to increase not only FSH but also LH effects on the ovaries acting through both exogenously administered and endogenous gonadotrophins (Davis and Rosenwaks, 1996; Fasouliotis et al., 2000; Tarlatzis et al., 2003). In spite of this, all patients had again the cycle cancelled because of poor follicular response.
Several strategies have been proposed to improve outcome in low responders but unfortunately all of these approaches have met with only limited success and to date, no compelling advantage for one stimulation protocol over another has been established (Keay et al., 1997; Surrey and Schoolcraft, 2000; Mahutte and Arici, 2002; Tarlatzis et al., 2003). Thus, after having unsuccessfully used one of the most popular of such strategies in the second ART attempt, we did not randomize our patients in their third ART cycle because we felt the previously published data were so conclusive that we would be committing our patients to a poor outcome if we proceeded with anyone of reported approaches focused to increase the number of follicles available for recruitment and development. On the other hand, one cannot ignore the fact that the differential ovarian response is a characteristic of individuals, one that, only to some extent, can be improved by manipulating the stimulation protocol. In the self-controlled case series method, all individual-level confounders are corrected for by virtue of the fact that individuals are matched to themselves. In fact, the use of different ovulation induction protocols in the same patient as previously reported (Couzinet et al., 1988; Shoham et al., 1991; Balasch et al., 1995) seems the more appropriate study design when ovarian performance and hormone concentrations but not pregnancy rate are to be compared (Daya, 1999). Therefore, in this setting, subjects in the current study constituted at best their own controls.
Other features deserve comment in the current investigation. First, most previous studies on the management of low responders included patients having an inadequate response in only one previous stimulation cycle and women who had had a peak E2 level as high as 1000 pg/ml or had produced even four oocytes in the previous cycle were characterized as low responders (Fasouliotis et al., 2000; Tarlatzis et al., 2003). In contrast, we included only patients having their first two ART attempts cancelled due to inadequate follicle recruitment. This is to be noted considering that we have recently reported that the history of an ART cycle cancelled due to poor follicular response in a standard stimulation protocol is a better predictor of cancellation in subsequent treatment cycles than age or FSH are. Notably, the poor ovarian response and ART outcome associated with previous cycle cancellation occur whatever the level of basal FSH (Peñarrubia et al., 2005b). On the other hand, the criteria for cycle cancellation used by us have been used in many other studies and seem to be the most widely applied criteria (Klinkert et al., 2004). Finally, among six pregnancies and five deliveries obtained in the twenty poor responders undergoing embryo transfer in this study, there were three pairs of twins. It has been reported that in all situations where more than four embryos were available for transfer, there was no greater birth rate for women receiving three embryos than for those receiving two, but there was a considerable increase in the rate of multiple births, and especially triplets (The ESHRE Capri Workshop Group, 2000). Remarkably, in the present study the mean number of embryos per patient obtained was <4 but the twin pregnancy rate was still high. This further stress the need to limit the number of embryos replaced to reduce the risk of multiple births and supports the idea that in most circumstances two embryos can be transferred without anticipating any significant diminution in the expected pregnancy or birth rates (The ESHRE Capri Workshop Group, 2000).
Patients experiencing a poor response in their first ART attempt but aged <41 years and having basal FSH level not elevated and thus considered as unexpected poor responders remains a challenge for the reproductive endocrinologist (Fasouliotis et al., 2000; Klinkert et al., 2004). Several mechanisms have been proposed to explain low response in these women such as the interference with FSH action due to low- and high-molecular weight proteins, the presence of antibodies against granulosa cells, defective angiogenesis, autocrine or paracrine alterations leading to decreased quantities of certain intraovarian peptides, FSH-receptor polymorphism, altered signal transduction after ligand binding protein, or low diffusion of exogenous gonadotrophins; however, none of these hypotheses has been definitely proven in low responders (Pellicer et al., 1998; Fasouliotis et al., 2000; Klinkert et al., 2004). Ultrasonographic studies, however, have shown that the population of follicles ready to be recruited during the initial follicular phase of a natural cycle is lower in such patients as compared with normal responders (Pellicer et al., 1998). This is in agreement with data derived from the current investigation. All patients included in the present study had basal FSH levels <10 IU/l which, according to previous studies by us where receiver operating characteristic (ROC) curve analysis was used (Creus et al., 2000), is the best threshold value for basal FSH in predicting ovarian response in ART cycles. In addition, the 25 patients included in this study had a mean (±SEM) basal antral follicle count (5.56 ± 0.49) significantly lower than that obtained among 50 patients having a completed first ART cycle (12.76 ± 1.30) (P < 0.0001) and who were randomly selected from our assisted reproduction program matching by race, age (±1 years), BMI (±1 kg/m2), basal FSH (±0.5 IU/l) and indication for ART (data not shown).
Finally, it should be noted that the present investigation included many very difficult responder patients who received, immediately preceding gonadotrophin therapy, a short-term low-dose testosterone treatment which was selected arbitrarily on the basis of a series of experiments in monkeys showing that androgens enhance follicular growth and survival (Vendola et al., 1998, 1999; Weil et al., 1998, 1999). In this regard, it is to note that it takes about three months for a given primordial follicle to reach the preovulatory stage (Gougeon, 1996). Because the time of exposure to testosterone was relatively reduced in our study, we hypothesize that it affects late events involved in follicular maturation rather than earlier stages. In support of this hypothesis are the findings that in primates the distribution of the androgen receptor expression is restricted to antral and late preantral follicles (Hillier et al., 1997; Weil et al., 1998, 1999). Furthermore, previous work suggests that androgens augment the expression of FSH receptors in granulosa cells (Weil et al., 1998, 1999). At present, it is not possible to determine whether androgens are rescuing follicles or simply increasing the number of recruited ones; further research is needed in this respect which may contribute to throw light on facts such as the lack of ovarian response in some testosterone treated patients in spite of having similar baseline antral follicle count to those reaching oocyte aspiration. On the other hand, the peak testosterone serum level reached in our study was about fourfold higher than the upper limit for normal reproductive age women in our laboratory and we do not know the potential deleterious effects of those high testosterone levels attained if used for a longer period than five days. Therefore, the daily dose, timing and duration of androgen supplementation may be critical to adequately stimulate folliculogenesis mainly considering a recent study in rhesus monkeys showing that chronic administration (for five days before and continuing throughout FSH and LH treatment) of high dose of androgens is antagonistic to gonadotrophin-stimulated ovarian function in primates (Zeleznik et al., 2004). It has been postulated that there is a threshold effect of androgens on follicular function such that antagonistic actions of androgens may be manifested at elevated concentrations. Also, concentration–dependent interactions between FSH and testosterone seem to exist, such that the ability of testosterone to interfere with FSH-stimulated ovarian function may be overridden by elevated levels of FSH (Zeleznik et al., 2004).
Therefore, the above-discussed aspects deserve further research mainly considering that in a preliminary report (Hugues et al., 2004), no beneficial effect of testosterone application on the ovarian response to FSH could be demonstrated in previous low responders. It should be noted, however, that there were some important methodological differences between the current investigation and that previous study (Hugues et al., 2004). Remarkably, in the preliminary report, low response was defined as peak E2 levels <1200 pg/ml and number of total retrieved oocytes 
5 in a previous cycle, patients had evidence for decreased ovarian reserve according to day 3 hormonal measurements, they were given either GnRH agonist or antagonist, and high dose testosterone (10 mg/day) during 15 days before FSH treatment was used in the IVF study cycle (Hugues et al., 2004). In contrast, in the current study, only patients having normal basal FSH levels and the first two IVF cycles cancelled because of poor follicular response in spite of vigorous gonadotrophin ovarian stimulation under pituitary suppression with the long protocol of GnRH agonist were included. In addition, the daily dose of testosterone was lower and the duration of androgen therapy shorter in our study.
It could be argued that the use of testosterone pretreatment implies a delay of initiation of FSH administration of five days and thus it could be possible that a change in antral follicle count could also have occurred without testosterone treatment. This seems unlikely, however, considering that it has been shown that GnRH agonist down-regulation does not significantly change antral follicle count both in patients with normal ovaries and in those with polycystic ovaries (Sharara et al., 1999; Hansen et al., 2003; Ng et al., 2004).
In conclusion, pretreatment with transdermal testosterone may be a useful approach for women known to be low responders on the basis of a poor response to controlled ovarian stimulation but having normal basal FSH concentrations. As many as 80% of patients having their first two IVF cycles cancelled because of poor follicular response in whom testosterone treatment was used in a third attempt, produced a fair number of oocytes, received two or three embryos per transfer and achieved an acceptable clinical pregnancy rate of 30% per oocyte retrieval. Further studies in a randomized setting are needed to confirm our findings and to establish the best timing and dosage of androgen therapy before widespread clinical use. In addition, whether the use of testosterone supplementation in patients with previous cycle cancellation associated with increased basal FSH levels can improve ovarian response in these women remains to be investigated.
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The authors thank Ms Paquita Antonell for her technical assistance. This work was supported in part by grants from the Instituto de Salud Carlos III (RCMN C03/08), and the Agència de Gestió d’Ajuts Universitaris i de Recerca-Generalitat de Catalunya 2005SGR 00573.
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Submitted on December 21, 2005; resubmitted on January 25, 2006; accepted on January 27, 2006.
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