Hum. Reprod. Advance Access originally published online on February 13, 2006
Human Reproduction 2006 21(5):1204-1211; doi:10.1093/humrep/dei481
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Effects of transdermal testosterone application on the ovarian response to FSH in poor responders undergoing assisted reproduction technique—a prospective, randomized, double-blind study
1 Reproductive Medicine Unit, Jean Verdier Hospital, University Paris XIII, Bondy, 2 Department of Hormonal Biochemistry, Hotel Dieu Hospital, Paris and 3 Department of Reproductive Biology, Jean Verdier Hospital, University Paris XIII, Bondy, France
4 To whom correspondence should be addressed at: Reproductive Medicine Unit, Jean Verdier Hospital, Avenue du 14 juillet, 93143 Bondy Cedex, France. E-mail: jean-noel.hugues{at}jvr.ap-hop-paris.fr
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Abstract |
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BACKGROUND: In primates, androgens can play a synergistic role with FSH in promoting the early follicular recruitment, which is critical in assisted reproduction technique programmes. OBJECTIVE: To assess whether poor responders can benefit from androgen application. METHODS: Inclusion criteria were a previous poor ovarian response to controlled ovarian stimulation and a decreased hormonal ovarian reserve. Selected women were randomized to receive either transdermal application of testosterone (n = 24) or placebo (n = 25) gel for 15 days before FSH treatment for a second IVF cycle. Similar GnRH analogue and equivalent FSH daily doses were used in both cycles. The primary outcome was the total number of oocytes retrieved. RESULTS: Testosterone gel application resulted in a significant increase in plasma testosterone levels but did not significantly improve the antral follicle count. Furthermore, after gel application, the main parameters of the ovarian response (numbers of pre-ovulatory follicles, total and mature oocytes and embryos) did not significantly differ between testosterone and placebo-treated patients. CONCLUSION: No significant beneficial effects of androgen administration on the ovarian response to FSH could be demonstrated. However, subsequent clinical trials are needed to determine whether an optimal dose and/or a longer duration of testosterone administration may be helpful.
Key words: androgen/ART/controlled ovarian stimulation/poor responders/testosterone
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Introduction |
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According to the two-cell/two-gonadotrophin theory, androgens play a critical role in ensuring adequate steroidogenesis (Ryan et al., 1968
). Indeed, androgens act as a substrate for the aromatase activity of the granulosa cells, where they are converted into estrogens. Furthermore, androgens may also exert a direct autocrine and/or paracrine effect to regulate follicular function. Specific androgen receptors that mediate the actions of these steroids have been identified by immunohistochemistry in the human ovary (Horie et al., 1992; Suzuki et al., 1994). It has also been shown that androgen receptors are developmentally regulated with a progressive disappearance, as the follicle proceeds to the final stage of maturation (Hillier et al., 1997).
The biological effects of androgens on the steroidogenic activity of granulosa cells have been extensively studied and include modulation of aromatase activity according to the stage of follicular development. Although a stimulatory effect is observed in small follicles, androgens negatively regulate aromatase activity in pre-ovulatory follicles (Harlow et al., 1988; Shaw et al., 1989). In contrast, the role of androgens in the process of folliculogenesis is still a matter of debate. Although androgen excess usually induces atresia of developing follicles in rodents (Hillier and Ross, 1979; Billig et al., 1993), they may exert a synergistic effect with FSH to promote follicular recruitment in primates. A short-term administration of high doses of androgens in monkeys induces dramatic morphological changes of the ovaries, which take on the appearance of polycystic ovaries (PCO) (Vendola et al., 1998): the total number of growing pre-antral and small antral follicles was increased by 2.5- to 4.5-fold, but the number of large antral follicles remained unchanged. The same group provided evidence for a positive effect of androgens on follicular proliferation, attested by a positive correlation between androgen receptor concentrations and expression of cell-proliferation markers and by a negative correlation between androgen receptor concentrations and the rate of cell apoptosis (Vendola et al., 1998; Weil et al., 1998). Moreover, androgens are likely to impact folliculogenesis in monkeys by increasing the number of FSH receptors expressed in granulosa cells (Weil et al., 1999). However, a recent study, using a more physiological model, in monkeys could not confirm that androgens improve the ovarian sensitivity to gonadotrophins (Zeleznik et al., 2004).
Besides these experimental data, further evidence for a potential positive effect of androgens on follicular proliferation and growth in humans emerged from pharmacological or pathological models. Firstly, a long-term exposure to large doses of exogenous testosterone in female-to-male transsexuals has been associated with morphological features of polycystic ovary syndrome (PCOS), with a significant increase in the number of small antral follicles (Spinder et al., 1989). Secondly, polycystic-like ovaries have been described in women with non-ovarian causes of hyperandrogenism, such as congenital adrenal hyperplasia and androgen-producing tumours. Lastly, high androgen production may account for the antral follicle excess usually observed in patients with PCOS (de Leo et al., 1998), as suggested by a highly significant positive correlation between the number of small follicles and androgen levels (Jonard et al., 2003; Pigny et al., 2003).
Altogether, these data suggest that, in non-human primates and in human beings, androgens play a critical role in the control of follicular development and in follicular sensitivity to FSH. Therefore, we assumed that androgen supplementation during the early phase of follicular recruitment might improve the number of small antral follicles as well as improve the ovarian sensibility to FSH. To test this hypothesis, a double-blind, prospective, randomized study was set up. This study assessed the effects of testosterone application on the ovarian response to FSH in a subgroup of patients who had previously experienced a poor ovarian response to controlled stimulation in relation to a low ovarian reserve.
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Subjects and methods |
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The design of the study was approved by the local ethics committee. All women signed an informed consent.
Patient characteristics
Women were enrolled if two simultaneous inclusion criteria were met: (i) a poor ovarian response to ovarian stimulation in a previous IVF or ICSI attempt, defined as plasma estradiol (E2) value <1200 pg/ml at HCG day and number of total retrieved oocytes 
5 and (ii) evidence of a decreased ovarian reserve, determined at day 3 of a spontaneous cycle and defined as plasma hormonal values (FSH, E2 or inhibin B) outside the normal range of the local standard (FSH > 12 IU/l, E2 > 70 pg/ml and inhibin B < 45 pg/ml). Exclusion criteria were women over the age of 42 years, history of previous ovarian surgery, ovarian endometriosis and endocrine and metabolic disorders.
Study design
This prospective, randomized, double-blind, placebo-controlled study was conducted in the Reproductive Medicine Unit, in Jean Verdier Hospital. The design was set up to perform a paired comparison of the ovarian parameters recorded in two consecutive cycles, each woman being used as her own control. The first cycle, defined as the control cycle (CC), allowed the selection of patients according to the inclusion criteria. The second cycle, performed after application of a gel containing testosterone or its identical placebo according to a randomization list, was defined as the treated cycle (TC).
Ovarian stimulation protocol
The protocol used for ovarian stimulation was comparable in the two consecutive cycles. The same GnRH analogue protocol as well as the same starting dose of recombinant FSH (Gonal-F®, Serono SA, Boulogne, France; Puregon, Organon, Puteaux, France) was applied for each woman in both the cycles. Cycles were monitored by hormonal determinations and ultrasound assessments. When an FSH dose adjustment was required in the CC, the equivalent adjustment was applied in the TC for each individual. In both cycles, 10 000 IU of urinary HCG (uHCG) (Gonadotrophines Chorioniques Endo, Organon) was administered when at least three follicles reached 17 mm in diameter. Oocyte retrieval was performed 36 h later for IVF or ICSI procedures. Up to three embryos were replaced after 2 or 3 days of in vitro culture. Luteal phase was supported by vaginal administration of natural progesterone 200 mg twice daily (Utrogestan, Besins International, Montrouge, France) and by injection of 2500 IU of uHCG (Gonadotrophines Chorioniques Endo) at day HCG +3, +6 and +9.
Transdermal testosterone application
Testosterone gel and its identical placebo gel were provided by Besins International in bottles fitted with metered pumps, allowing a daily dosing of 1 g of gel. Testosterone gel is a colourless hydroalcoholic, 1% testosterone gel. Women applied once-daily 1 g of gel (10 mg of testosterone) on the external side of the thigh. Testosterone absorption with the gel is approximately 10%. Pharmacokinetic studies have previously shown that the transdermal route allows for delivering testosterone in a more physiological way than the oral administration, with a steady concentration of plasma testosterone for 24 h following application (Slater et al., 2001). The objective was to achieve a plasma testosterone concentration within the upper quartile of the normal range for women. For randomization, the allocation sequence was generated by a random permutation table (blocks of four). Women were enrolled by only one physician (N.M.), who assigned consecutive numbers to women, by order of inclusion.
Either testosterone or placebo gels were applied for 15–20 days in the period preceding the second stimulation for IVF or ICSI, i.e. during the period of pituitary desensitization in women treated with a long GnRH agonist protocol or during pill administration in women treated with another analogue protocol.
Evaluation procedures and outcome measures
The primary endpoint was the total number of retrieved oocytes. The secondary endpoints were the number of developing follicles, the plasma E2 levels at HCG day, the total FSH dose, the numbers of metaphase II (MII) oocytes and embryos and the cancellation rate. In addition, the pregnancy rate per oocyte retrieval, the implantation rate per embryo (ratio of number of gestational sacs to number of embryos transferred) and the birth rate were recorded and analysed.
Vaginal ultrasonography
Ultrasonography was performed with a 6-MHz vaginal sonde using Toshiba SSA-340A by the same physician (I.C.-D.), who was not informed of patients’ treatment allocation. Ultrasound assessments were done at days 2–4 of the cycle before gel administration, on the last day of gel application and from the FSH administration to the day of HCG administration.
Hormonal measurements
Measurements of plasma FSH, LH, E2 and progesterone values were carried out using commercially available chemoluminescence immunoassays with automated Elecsys immunoanalyser (ECLIA, Roche Diagnostic, Meylan, France). The sensitivity of the assays for FSH and LH was 0.1 IU/l, with intra- and inter-assay coefficients of variation (CV) between 3% and 6% and between 3% and 4%, respectively. The sensitivities of the assays for E2 and progesterone were 5 pg/ml and 0.03 ng/ml, with intra- and inter-assay CV between 5% and 10% and between 3% and 5%, respectively. Measurements of plasma testosterone and extracted 
4-androstenedione values were carried out by radioimmunoassays (DSL4000, DSL France and Immunotech Beckman-Coulter, France, respectively). The sensitivity of the assays for testosterone and 4-androstenedione was 0.1 ng/ml, with intra- and inter-assay CV between 9.1% and 9.6% and between 8.1% and 11.9%, respectively. Measurements of plasma inhibin B and antimüllerian hormone (AMH) (also known as müllerian-inhibiting substance) or values were carried out by enzyme-linked immunosorbent assay (ELISA) (Oxford-BioInnovation, Argène Biosoft and Immunotech Beckman-Coulter, France, respectively). The sensitivities of the assays for inhibin B and AMH were 10 pg/ml and 0.2 ng/ml, with intra- and inter-assay CV between 13.5% and 21.5% and between 12.3% and 14.2%, respectively.
Sample size
The number of patients to be enrolled in this study was calculated for a paired analysis comparing the CC and the TC. Previous analysis of data from cycles performed in poor responders has shown that the mean number of total oocytes (±SD) was 3 (±5.06) and variance 
= 2.25. Assuming that testosterone gel application might improve the number of total oocytes by a factor of 2, the number of patients to be included was estimated at 28 in each group, on the basis of a unilateral test with a power at 95% and 
error at 5%.
Statistical analysis
Results are expressed as mean ± SD and with 95% confidence interval. Statistical analysis was performed using StatView 4.5 (Abacus concept software). A paired per protocol analysis was performed. Means were compared with repeated measures analysis of variance (ANOVA) and nominal variables compared with paired chi-square test. A P-value <0.05 was considered as statistically significant.
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Results |
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TopAbstractIntroductionSubjects and methodsResultsDiscussionAcknowledgementsReferences |
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Among 62 women eligible for inclusion criteria, 53 women met the inclusion criteria and were enrolled in the study between October 2001 and December 2003. The difference between the scheduled and the actual numbers of enrolled patients was related to some difficulties that we faced to recruit women who met the strict inclusion criteria. As shown in the flow diagram designed according to the Consolidated Standards of Reporting Trials (CONSORT) statement (Moher et al., 2001
) (Figure 1), 26 patients were allocated to placebo and 27 to testosterone gel application. In each group, one patient stopped treatment for personal reasons. In the testosterone-treated group, two patients were discontinued by investigator decision: one due to a spontaneous pregnancy and one due to the absence of ovarian desensitization. Therefore, 25 patients of the placebo group and 24 patients of the testosterone group were included in the final analysis.
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Baseline characteristics of women
As summarized in Table I, the mean age, BMI, the type of infertility and the proportion of IVF or ICSI cycles were comparable in both groups. The duration of infertility was longer in the treatment group (P 0.006). The imbalance in this latter criterion was unexpected. However, we consider that the two groups are comparable because it is generally accepted that any difference in the duration of infertility would not impact the primary outcome, which is the total number of retrieved oocytes. Both groups displayed comparable evidence of a low ovarian reserve, assessed by day 3 hormonal values, and low ovarian response to FSH, attested by the need for high doses of FSH, low E2 levels on the day of HCG and a low number of retrieved oocytes and transferred embryos (Table I).
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Effects of gel application on basal hormonal levels and antral follicles count (Figure 2)
The comparative analysis of the mean plasma testosterone values at day 3 (D3) and at the end of gel application (S1) showed a significant increase from 0.58 ± 0.16 to 1.55 ± 0.89 ng/ml (P < 0.0001) in testosterone-treated women, whose plasma testosterone levels were above the normal range of an age-matched women control group (Figure 2). In contrast, no significant change in plasma testosterone concentrations was observed in women who received the placebo. Therefore, pituitary down-regulation had no impact on the plasma testosterone level. Similarly, plasma AMH levels were not significantly affected by either testosterone or placebo gel administration. Furthermore, the count of small antral follicles (3–9 mm in diameter) was not significantly modified by gel application in both testosterone- and placebo-treated patients. A comparative analysis of the number of follicles according to their size (
3–5 mm and 6–9 mm in diameter) could not show any significant difference between the two treatment groups.
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Effects of gel application on the ovarian sensitivity to FSH and on the IVF outcome
The effects of gel application were firstly analysed by comparing the ovarian parameters recorded in testosterone and placebo TCs. As shown in Figure 3, the comparative analysis of the number of follicles at day 1, day 8 and HCG day of the FSH stimulation did not show any significant difference between both treated groups. Indeed, the number of small (<10 mm) follicles similarly declined during FSH stimulation, whereas the numbers of medium-sized (10–14 mm) and large (>14 mm) follicles gradually increased throughout FSH stimulation, without any significant difference between the two groups. Criteria for HCG administration were met, and oocyte retrievals were performed in 16 women treated with testosterone (67%) and in 20 women treated with placebo (80%) (P = 0.45).
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Table II summarizes the total dose of FSH used during the ovarian stimulation, the plasma E2 values at the time of HCG and the number of follicles, retrieved oocytes and total embryos in the CCs and in the placebo or testosterone TCs. The overall ANOVA paired analysis could not demonstrate any significant difference.
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A subsequent paired comparison was performed between the ovarian parameters of the CCs and TCs. As shown in Figure 4, there was a trend towards an increase in the number of follicles, oocytes and embryos in both CCs and TCs. The difference was significant only for the number of total oocytes. In addition, the difference in MII oocytes was significant only in the testosterone group. However, the comparison between the number of follicles, oocytes and embryos observed in placebo- and testosterone-treated patients did not show any significant difference.
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P = 0.059 with mean paired comparison between CC and TC in each group.The clinical pregnancy rate and implantation rate are reported in Table II. Although one and four clinical pregnancies were observed in placebo- and testosterone-treated patients, respectively, the low number of patients enrolled in each group precluded any statistical comparison.
Gel administration and adverse effects
All women reported that gel was easy to apply, and no significant adverse effect, systemic or on application site, was reported.
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Discussion |
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This prospective, randomized, double-blind, placebo-controlled study was not able to demonstrate any beneficial effect of testosterone application on the ovarian response to FSH in women who had previously experienced a poor ovarian response. Indeed, increasing testosterone plasma levels before FSH treatment was not effective at improving the IVF outcome; however, we cannot exclude the possibility that some subtle improvements went undetected in those patients treated with optimized large dose of FSH because of a poor ovarian reserve.
Although both FSH and LH play a critical role in folliculogenesis, the regulation of this process by steroids is still unknown. Recent experiments performed in monkeys allowed to conclude that androgens may exert a positive effect on the process of follicular growth (Vendola et al., 1998; Weil et al., 1999). Conclusions from these studies are in line with other observations in humans in whom administration of high doses of testosterone induced the appearance of PCOs (Amirikia et al., 1986; Futterweit and Deligdisch, 1986; Pache et al., 1991), with a significant increase in the number of small antral follicles (Spinder et al., 1989). However, Zeleznik et al. (2004), using a gonadotrophin clamp model, could not confirm any positive effect of testosterone and dihydrotestosterone on follicular function in rhesus monkeys. Moreover, the impact of these findings is limited by the small number of tested animals and by the huge variability in the E2 secretory patterns observed between animals.
Studies performed in human beings and using physiological androgen supplementation are still scarce. Casson et al. (2000) assessed the effects of dehydroepiandrosterone (DHEA), a steroid precursor, on the ovarian responsiveness to FSH. For that purpose, DHEA (80 mg/day) was administered 2 months before ovarian stimulation and intrauterine insemination to patients who had previously experienced a poor response to gonadotrophins. In these patients, whose baseline serum dehydroepiandrosterone sulphate (DHEA-S) concentrations were relatively low, the authors observed an increase in testosterone serum levels and an improvement in the hormonal response to gonadotrophins. Although the small number of patients enrolled in this study as well as the absence of randomization and placebo-controlled group precludes any firm conclusion, this study was the first to address the issue of the potential benefit of androgen supplementation on the ovarian sensitivity to FSH in 35- to 40-year-old women with a low sensitivity to FSH.
Moreover, it has been reported that plasma androgen concentrations decrease in elderly women in relation to an age-related decline in ovarian androgen secretion (Piltonen et al., 2003). Indeed, there is some evidence that ageing ovary is no more an androgen-producing gland (Couzinet et al., 2001). This could be related to the decreased responsiveness to LH stimulation reported during the late thirties (Piltonen et al., 2003). Furthermore, during the menopausal transition, the ovarian sensitivity to FSH is reduced and the IVF success is usually poor. In clinical practice, the ovarian reserve is commonly assessed by measurement of hormonal parameters such as FSH, E2 and inhibin B, which only reflect the healthy condition of granulosa cell. Until recently, little attention has been paid to the assessment of the theca cell function in ageing women to predict the ovarian responsiveness to gonadotrophins. However, Frattarelli and Peterson (2004) recently reported interesting data on the predictive value of serum androgen levels in 43 women enrolled in an IVF programme. The purpose of this study was to investigate whether serum testosterone levels may be correlated with IVF outcomes. Although serum androgen concentrations did not correlate with the number of antral follicles, these authors reported a negative correlation between serum androgen levels and the total dose of FSH or the days of stimulation. More important, they observed that a low baseline testosterone value is predictive of a poor cycle outcome. Indeed, in this study, women with baseline testosterone value lower than 0.2 ng/ml were five times less likely to achieve pregnancy. Similarly, Barbieri et al. (2005) recently reported a positive correlation between baseline plasma testosterone levels and plasma E2 values at HCG day or the number of retrieved oocytes in regularly cycling infertile women undergoing their first IVF cycle.
Therefore, these data provide new evidence that assessment of androgen secretion may be useful to predict the ability of the ovary to adequately respond to gonadotrophins. Moreover, they also suggest that a subgroup of women with low serum androgen levels may benefit from androgen supplementation to improve the ovarian sensitivity to gonadotrophins.
The absence of any beneficial effects of testosterone application observed in our study may be due to several reasons. Firstly, the androgen secretion of women enrolled in our IVF programme was presumably normal. Indeed, their basal plasma androgen concentrations were within the normal range. Therefore, it may be assumed that androgen supplementation may be effective only in patients with a decreased androgen production. If this hypothesis is valid, then this would imply that basal androgen secretion should be more carefully assessed, particularly in patients with a low ovarian response, whatever the ovarian reserve. Our data also show that basal testosterone concentration may be still within the normal range even in patients with a poor ovarian reserve. However, it should be emphasized that no simple assay of total testosterone has been shown to produce reliable results in women with low testosterone levels (Rivera-Woll et al., 2004). Further investigation is required to assess whether dynamic tests of theca cell stimulation would helpful in assessing the ovarian sensitivity to FSH.
Secondly, the timing and the duration of androgen supplementation may be critical in adequately stimulating follicular growth. In our study, testosterone was only applied before FSH administration, and the application was restricted to 2 weeks to prevent any side effects. However, as suggested by the data reported by Casson et al. (2000), a longer duration of androgen application before and during FSH stimulation might be required to effectively improve follicular growth.
Moreover, the total amount of androgen supplementation may be also a major determining factor for success. In our study, the daily dose and the route of administration were chosen to get steady supraphysiological serum testosterone concentrations. Such a design largely differs from those reported in monkey experiments or in studies performed in female-to-male transsexuals. Till now, data on the dose–response effects of androgen are not available, and further clinical studies are clearly needed.
Lastly, to validate the concept of the androgen control of follicular growth, a systemic administration of androgens might not be optimal. Indeed, in situ androgen concentration is likely to be critical for achieving a paracrine stimulatory effect on follicles. Moreover, intraovarian excess of androgens in patients with PCOS is presumably responsible for the proliferation of small antral follicles. In that respect, an alternative to systemic androgen administration could be the administration of exogenous LH or LH-like activity to stimulate in situ production of androgens. A recent report did suggest that LH supplementation may be beneficial for women with low ovarian response to FSH (De Placido et al., 2005).
Therefore, this study allows to suggest that testosterone supplementation before FSH treatment in low responders is not effective at improving either the number of small antral follicles or the ovarian sensitivity to FSH. However, these results do not exclude any impact of androgens on folliculogenesis. Indeed, the dose and the duration of testosterone administration might have been suboptimal to adequately stimulate folliculogenesis and androgen treatment could be more effective in selected patients with low basal levels of androgens and/or in select patients with poor ovarian response without poor ovarian reserve. Finally, systemic administration of testosterone may not be adequate to ensure an optimal concentration of intraovarian androgens. One therapeutic alternative might be to stimulate androgen synthesis through LH administration, and further studies are required to validate this hypothesis.
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Acknowledgements |
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We are grateful to E. Le Nestour, V Masini-Esteve and Besins International for providing Testogel and placebo gel as well as for their technical support.
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Submitted on August 31, 2005; resubmitted on October 25, 2005; accepted on December 6, 2005.
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