Over the last decade, an increasing interest has focused on the role of androgen supplementation in women undergoing in-vitro fertilization (IVF) treatment, especially in women considered as poor ovarian responders (POR) who exhibit a lower response than expected to controlled ovarian stimulation (COS). Importantly, POR affects a significant proportion of infertile couples seeking fertility advice. Given its high incidence (9–24% of women attending an IVF unit  and up to 50% when older than 40 years ), the pursuit for new therapies to help this subset of patients is an ongoing quest in search for answers.
Numerous treatment approaches have been evaluated as an option to address the problem of POR, including mild stimulation protocols, alternative stimulation schemes, high gonadotropin doses, natural cycle IVF, or adjuvant therapies, among others. However, despite the vast amount of studies conducted, no ‘optimal’ protocol seems to change the prognosis for these patients .
Adjuvant therapies, such as androgen supplementation, have been considered as an option to increase the recruitable follicular pool in this population with an aim to increase the number of oocytes retrieved, and thus pregnancy outcomes. However, despite the several studies reporting promising results in favor of the use of androgens in women with POR, recent accumulating evidence does not appear to follow the initial promising results with all types of androgen pretreatment [4–6,7▪].
The aim of the current review is to provide an overview of the androgen production and metabolism in women, their role in the process of folliculogenesis and whether evidence support that all or some androgens may play any role in increasing the functional ovarian reserve in women with POR, and therefore promote more recruitable antral follicles, to allow the initiation of COS and induce an increase in the number of retrieved oocytes.
ANDROGEN PRODUCTION AND METABOLISM IN WOMEN
The primary androgen circulating in women, in serum concentration descending order, are dehydroepiandrosterone sulfate (DHEAS), dehydroepiandrosterone (DHEA), androstenedione, testosterone, and dihydrotestosterone (DHT) [8,9,10▪▪,11]. Yet, only testosterone and DHT (an androgen 3–10 times more potent than testosterone) bind directly to the androgen receptor, whereas the others are androgen precursors that require conversion to testosterone and DHT to exert androgenic effects [8,11], through the conversion into estrogens, which exert indirect actions via the estrogen receptor [10▪▪,12▪].
Dehydroepiandrosterone vs. testosterone production in women
Dehydroepiandrosterone is produced by the adrenal zona reticularis (50%), 20% from the stromal tissue from the ovary's theca cells, and 30% is derived from circulating DHEAS [8,13]. The production rate is 6–8 mg/day and circulating concentrations are between 1 and 10 ng/ml. Dehydroepiandrosterone can also be produced intracellularly from DHEAS in the course of peripheral androgen synthesis .
On the other hand, testosterone's production rate in the typical female is around 0.2–0.3 mg/day, and circulating levels are in the range 0.2–0.7 ng/ml. Approximately 50% arises from peripheral conversion of androstenedione (and a small amount from DHEA) to testosterone, whereas the ovary secretes 25%, and 25% by the adrenal glands’ zona fasciculata. Testosterone is carried in peripheral blood bound to sex steroid hormone-binding globulin (SHBG) .
Androgens in women and age
Overall, serum androgen levels have been reported to decline with age from the early reproductive years. They do not vary with the menopause transition, and, except for the preandrogen DHEAS, increase slightly during the seventh decade .
ANDROGEN'S ROLE IN FOLLICULOGENESIS
The role of androgens on follicle development has been investigated in several early animal studies. Although 25 years ago, androgens were suspected of increasing follicle atresia  and were considered potentially detrimental to normal folliculogenesis, owing to the poor oocyte quality observed in human hyperandrogenic polycystic ovary syndrome (PCOS) , androgens are hormones playing a crucial role in the process of folliculogenesis. Despite that estrogens are the hormones considered to be the critically associated with folliculogenesis and female reproduction , accumulating evidence suggests that androgens do also appear to play a crucial role in steroidogenesis, follicular growth, and development. Androgens action on the female ovary is mediated either indirectly by their conversion to estrogens and 3ß-diol, which can activate the estrogen receptors or via a direct action on the androgen receptor [10▪▪].
Evidence that the androgen receptor has been conserved throughout evolution in all mammalian species confirms its importance in ovarian physiology [10▪▪]. In fact, the androgen receptor is expressed everywhere along the reproductive axis (hypothalamus–pituitary–gonads), including brain, ovaries (stroma, follicles and, corpora lutea) and, even more, androgen receptors are expressed through almost every stage of follicular development showing well defined patterns depending on the different follicular developmental stages [10▪▪,11].
Androgens appear to play a role in the primordial follicle initiation phase, a role, which may not be directly associated through an action via the androgen receptor. Despite evidence in the mouse and primates of stimulation of initiation by testosterone and DHT, there has been no evidence of androgen receptor expression in the primordial follicle, which would suggest that androgen action would be mediated via indirect paracrine mechanisms .
However, this is not the case during the development from preantral to the antral stage, in which testosterone appears to have an action on folliculogenesis mediated via the androgen receptor. In-vitro culture of mouse preantral follicles in the presence of antiandrogen antibodies or an androgen receptor antagonist (bicalutamide) significantly suppressed follicle growth , whereas treatment with DHT restored follicular growth . These stimulatory effects appear not to be because of aromatization because the addition of an androgen receptor antagonist (flutamide) blocked growth and addition of estrogens alone or an aromatase inhibitor (fadrozole) did not affect growth .
On the other hand, the role of androgens in the late follicular development stages is not that clear. Administration of testosterone or DHT during preovulatory follicle development, oocyte maturation, and ovulation did not increase preovulatory follicle numbers in primate ovaries . However, a dose-dependent response was observed in mice by improving the ovulatory response to superovulation [11,22]. Collectively, these data suggest that optimal levels of androgens are needed to maintain normal ovulatory function.
Androgen receptor knockout mouse models
Recent animal experiments in androgen receptor knockout (ARKO) mice elucidated how fertility impairment appears to be primarily ovarian . By generating granulosa cell-specific and oocyte-specific ARKO mice, researchers asserted where androgen-dependent effects were located , concluding that almost all reproductive phenotypes observed in global ARKO mice proved explainable by androgen receptor expressions in granulosa cells, including premature ovarian failure (POF), subfertility with longer estrous cycles and fewer ovulated oocytes, more preantral and atretic follicles, fewer antral follicles and fewer corpora lutea. Additionally, more specific ARKO models have been designed with precise deletions to reproduce the different roles of androgen actions along the developmental stages. These ARKO models include: granulosa cells (GCARKO) , theca cells (TCARKO) , oocyte (OoARKO) , pituitary (PitARKO) , and neurons (NeurARKO) . In the same direction, studies in primates showed that testosterone supplementation significantly increases the number of primary preantral and small antral follicles  in a dose and duration of administration-dependent manner .
Additional evidence of a role of androgen receptor actions in ovarian function comes from in-vitro studies, where androgens, have been published to promote follicle growing and development [19,20], with stimulatory effects blocked by a nonsteroidal androgen receptor blocker (antagonist; bicalutamide) . Still, contrasting conclusions have been reported concerning plausible androgen effects on ovarian function with uncertainty arising from the conversion of androgens into estrogens, which exert indirect effects via the estrogen receptor. Additionally, because, rather than pure blockers, androgen receptor antagonists frequently have both agonist and antagonist properties.
ANDROGENS IN OVARIAN STIMULATION
Off-label use of androgens in ovarian stimulation
The use of DHEA and testosterone in IVF increased in the past few years, as it is thought they may increase the chances of conception in women with POR . A recent survey by our group has shown that more than 40% of physicians in Europe and Australia are using off-label androgens in women with POR [29▪]. Still, their use remains controversial because of a lack of robust evidence for their efficacy and, more importantly, their safety.
Up to date, the vast majority of the initial studies in humans did not focus on the use of testosterone but the use of DHEA. Several cohort and case–control studies using DHEA demonstrated promising results in POR undergoing IVF; however, recent well designed randomized controlled trials (RCTs) failed to identify any significant improvement in ovarian reserve markers and ovarian response to stimulation in these women  (Table 1). This is attributed to the distinct mechanism of action between DHEA and testosterone and their affinity with the androgen receptor. Although DHEA functions as an endogenous precursor to more potent androgens, such as testosterone and DHT, DHEA is only a weak partial agonist of the androgen receptor . In this context, and because of competition for binding with full agonists like testosterone, it can behave more like an antagonist depending on circulating testosterone and dihydrotestosterone (DHT) levels, and hence, like an antiandrogen .
Furthermore, this is even more evident if we consider a recent RCT, which analyzed intrafollicular DHEA concentrations following administration of DHEA or placebo [7▪], in which, although DHEA increased intrafollicular levels of DHEA-S as compared with placebo, there were no significant differences in follicular testosterone levels measured between control and DHEA group [7▪].
Contrary to DHEA, testosterone is an androgen with a high affinity with androgen receptor that directly binds to the receptor. A systematic review of the literature identified previous small RCTs to increase the reproductive outcomes of IVF patients [32–34] (Table 1).
Nevertheless, a significant shortcoming in most of these published studies is the lack of consistency in the mode and duration of administration. Transdermal testosterone has been used in most of the studies in relatively high doses before ovarian stimulation with a duration ranging from 5 to 21 days [33–36]. Nonetheless, this might not be consistent with the folliculogenesis process in humans, given that the transition from the preantral to antral follicles lasts approximately 65–85 days [37,38]. Assuming that early research has shown that androgen receptors are present in the early follicular development stages in secondary or even primary follicles  and that testosterone in primates can augment the population of preantral follicles [18,21], one could speculate that a duration of administration of only 21 days prior to IVF would not be enough to increase the recruitable cohort of follicles.
On the contrary, administration of testosterone for 2 months might be the proper plan for PORs, given that only in such a condition, the duration of administration would be enough in order to effectively enhance the number of preantral follicles that reach the antral stage. In this context, androgen administration might increase the susceptibility of the antral follicle to improve follicle recruitment through exogenous gonadotropins.
On the other hand, doses as high as 12.5 mg per day, that have been adopted in the past [33–35], may not be the proper dose needed. Although previous studies have shown that high doses may affect the number of oocytes , others failed to demonstrate any impact [34,35]. However, what is very important to underscore is that with such high doses, especially in premenopausal women, who still produce endogenous testosterone, we might significantly exceed the normal high ranges of serum testosterone levels in women and we may even approach male testosterone levels, potentially causing an iatrogenic hyperandrogenemia and consequently diminished oocyte developmental competence .
Considering that pharmacokinetic studies in postmenopausal women have shown that exogenous administration of 13.2 mg testosterone per day in combination with estrogens may result in serum testosterone levels, which could exceed 100 ng/dl, we need to be extremely cautious before considering high doses in women with POR. The reason is, not only the fact that those serum levels may induce a high incidence of androgenic side effects, but also that we are unaware whether this may induce a detrimental instead of a beneficial effect on patients’ ovarian reserve. The same situation presents behind the adverse effects of very high testosterone levels on ovarian reserve markers seen in female to male transgenders [40,41]. Consequently, and based on testosterone pharmacokinetics, it can be hypothesized that daily testosterone dose of 5.5 mg may be the essential daily dose, if any benefit is to be anticipated, given that such a dosing scheme is more likely to induce serum levels reaching the high normal premenopausal testosterone levels.
Taking all these considerations into account, an Investigators Group developed the T-TRANSPORT trial [42▪].
Entirely in line with evidence we have learned from ovarian physiology and the testosterone pharmacokinetics, our group is currently conducting one of the largest investigator-initiated studies concerning the use of transdermal testosterone gel in women with POR, the Testosterone TRANSdermal Gel for Poor Ovarian Responders Trial (T-TRANSPORT; NCT02418572 available at http://clinicaltrials.gov), which includes more than five IVF centers in at least four European countries.
T-TRANSPORT is a double-blind placebo-controlled randomized controlled trial, with suitable sample size, including PORs fulfilling ‘Bologna criteria’ developed by the ESHRE working group on Poor Ovarian Response Definition . The aim of the trial is to test the effect of administration of transdermal testosterone for 2 months prior to ovarian stimulation under a long agonist protocol, in line with previous work indicating that 2 months androgen pretreatment may equip preantral follicles with more FSH receptors and increase the cohort of follicles surviving to the recruitable antral stage [36,43].
The daily dose of transdermal testosterone gel (TTG) in the T-TRANSPORT is 0.55 g (5.5 mg testosterone/day). The specific dose was selected based on previous pharmacokinetic studies in women according to which daily application of 5 mg of transdermal testosterone cream  or TTG [45,46] is likely to restore free testosterone levels to the premenopausal reference range.
Although no side effects had been described after pretreatment with higher doses of 12.5 mg TTG for 21 days in a previous randomized controlled trial , that high testosterone doses result in supraphysiologic total and free testosterone levels. Therefore, the dose of 0.55 g TTG (5.5 mg testosterone/day) has been selected for the T-TRANSPORT trial as this will restore total and free testosterone levels to levels above and within the upper normal reference range.
The crucial differences from the trials performed until now is not only the testosterone dose (5.5 mg/d) and the duration of administration of testosterone, which exceeds 60 days, but also the robust study design with fixed stimulation protocols, the double-blind placebo-controlled setup, and the large sample size outnumbering all previous trials cumulatively, with an adequate power to detect significant differences in pregnancy rates between the randomized groups.
In conclusion, the available body of evidence from clinical studies is limited in order to drive definitive conclusions on the role of androgens in women with POR, mainly because the short administration and the high dose of testosterone are not in line with the ovarian actions of androgens and the presence of androgen receptors during follicular development. Consequently, results from the ongoing T-TRANSPORT trial are essential to obtain conclusive evidence on whether transdermal testosterone gel can improve the reproductive outcome in poor ovarian responders.
Financial support and sponsorship
Conflicts of interest
N.P.P. received unrestricted grants from BESINS healthcare, Ferring Pharmaceuticals and Roche Diagnostics associated with TTRANSPORT trial. N.P.P. has received honoraria for lectures and consultancy feed from BESINS healthcare, Ferring Pharmaceuticals, and Roche Diagnostics.
REFERENCES AND RECOMMENDED READING
Papers of particular interest, published within the annual period of review, have been highlighted as:
- ▪ of special interest
- ▪▪ of outstanding interest
1. Ubaldi FM, Rienzi L, Ferrero S, et al. Management of poor responders in IVF. Reprod Biomed Online
2. Ferraretti AP, La Marca A, Fauser BCJM, et al. on behalf of the ESHRE working group on Poor Ovarian Response Definition. ESHRE consensus on the definition of ‘poor response’ to ovarian stimulation for in vitro fertilization: the Bologna criteria. Hum Reprod
3. Polyzos NP, Nwoye M, Corona R, et al. Live birth rates in Bologna poor responders treated with ovarian stimulation for IVF/ICSI. Reprod Biomed Online
4. Yeung TWY, Li RHW, Lee VCY, et al. A randomized double-blinded placebo-controlled trial on the effect of dehydroepiandrosterone
for 16 weeks on ovarian response markers in women with primary ovarian insufficiency. J Clin Endocrinol Metab
5. Yeung TWY, Chai J, Li RHW, et al. A randomized, controlled, pilot trial on the effect of dehydroepiandrosterone
on ovarian response markers, ovarian response, and in vitro fertilization outcomes in poor responders. Fertil Steril
6. Yeung T, Chai J, Li R, et al. A double-blind randomised controlled trial on the effect of dehydroepiandrosterone
on ovarian reserve markers, ovarian response and number of oocytes in anticipated normal ovarian responders. BJOG Int J Obstet Gynaecol
7▪. Narkwichean A, Maalouf W, Baumgarten M, et al. Efficacy of Dehydroepiandrosterone
(DHEA) to overcome the effect of ovarian ageing (DITTO): a proof of principle double blinded randomized placebo controlled trial. Eur J Obstet Gynecol Reprod Biol
Despite the results shown by other studies, these authors show that DHEA supplementation before stimulation does not seem to improve reproductive outcomes (number of recovered oocytes, clinical pregnancy rates, and live birth rates) in women with predicted poor ovarian response.
8. Burger HG. Androgen
production in women. Fertil Steril
2002; 77 (Suppl 4):S3–5.
9. Davison SL, Davis SR. Androgens in women. J Steroid Biochem Mol Biol
10▪▪. Walters KA, Handelsman DJ. Role of androgens in the ovary. Mol Cell Endocrinol
This outstanding review summarizes the conclusions from clinical, animal, novel ARKO mouse models, and pharmacological studies to provide an understanding of the critical role of the androgens in the ovary. Also provides insights into its implications in humans.
11. Walters KA. Role of androgens in normal and pathological ovarian function. Reproduction
12▪. Franks S, Hardy K. Androgen
action in the ovary. Front Endocrinol
This excellent up-to-date review provides evidence that androgens have a clear and fundamental physiological role in follicle development, and in estrogen production by antral follicles.
13. Longcope C. Adrenal and gonadal androgen
secretion in normal females. Clin Endocrinol Metab
14. Davison SL, Bell R, Donath S, et al. Androgen
levels in adult females: changes with age, menopause, and oophorectomy. J Clin Endocrinol Metab
15. Hsueh AJW, Billig H, Tsafriri A. Ovarian follicle atresia: a hormonally controlled apoptotic process*. Endocr Rev
16. Qiao J, Feng HL. Extra- and intra-ovarian factors in polycystic ovary syndrome: impact on oocyte maturation and embryo developmental competence. Hum Reprod Update
17. Dewailly D, Robin G, Peigne M, et al. Interactions between androgens, FSH, anti-Müllerian hormone and estradiol during folliculogenesis in the human normal and polycystic ovary. Hum Reprod Update
18. Vendola K, Zhou J, Wang J, et al. Androgens promote oocyte insulin-like growth factor I expression and initiation of follicle development in the primate ovary. Biol Reprod
19. Murray AA, Gosden RG, Allison V, Spears N. Effect of androgens on the development of mouse follicles growing in vitro. Reproduction
20. Wang H, Andoh K, Hagiwara H, et al. Effect of adrenal and ovarian androgens on type 4 follicles unresponsive to FSH in immature mice. Endocrinology
21. Vendola KA, Zhou J, Adesanya OO, et al. Androgens stimulate early stages of follicular growth in the primate ovary. J Clin Invest
22. Ware VC. The role of androgens in follicular development in the ovary. I. A quantitative analysis of oocyte ovulation. J Exp Zool
23. Sen A, Hammes SR. Granulosa cell-specific androgen
receptors are critical regulators of ovarian development and function. Mol Endocrinol
24. Walters KA, Middleton LJ, Joseph SR, et al. Targeted loss of androgen
receptor signaling in murine granulosa cells of preantral and antral follicles causes female subfertility1. Biol Reprod
25. Ma Y, Andrisse S, Chen Y, et al. Androgen
receptor in the ovary theca cells plays a critical role in androgen
-induced reproductive dysfunction. Endocrinology
26. Wu S, Chen Y, Fajobi T, et al. Conditional knockout of the androgen
receptor in gonadotropes reveals crucial roles for androgen
in gonadotropin synthesis and surge in female mice. Mol Endocrinol
27. Caldwell ASL, Edwards MC, Desai R, et al. Neuroendocrine androgen
action is a key extraovarian mediator in the development of polycystic ovary syndrome. Proc Natl Acad Sci USA
28. Nagels HE, Rishworth JR, Siristatidis CS, Kroon B. Androgens (dehydroepiandrosterone
) for women undergoing assisted reproduction. Cochrane Database Syst Rev
29▪. Andersen MF, Drakopoulos P, Humaidan P, et al. Off-label use of androgens and letrozole in infertile women - a multinational survey in Europe and Australia. Hum Reprod
This abstract from the 34th ESHRE meeting shows that clinicians very often recommend off-label androgens in reproductive medicine even when not always robust evidence is in favor of the treatment.
30. Gao W, Bohl CE, Dalton JT. Chemistry and structural biology of androgen
receptor. Chem Rev
31. Clark BJ, Prough RA, Klinge CM. Mechanisms of action of dehydroepiandrosterone
. in vitamins and hormones. Elsevier; 2018:29–73.
32. González-Comadran M, Durán M, Solà I, et al. Effects of transdermal testosterone
in poor responders undergoing IVF: systematic review and meta-analysis. Reprod Biomed Online
33. Kim C-H, Howles CM, Lee H-A. The effect of transdermal testosterone
gel pretreatment on controlled ovarian stimulation and IVF outcome in low responders. Fertil Steril
34. Massin N, Cedrin-Durnerin I, Coussieu C, et al. Effects of transdermal testosterone
application on the ovarian response to FSH in poor responders undergoing assisted reproduction technique—a prospective, randomized, double-blind study. Hum Reprod
35. Bosdou JK, Venetis CA, Dafopoulos K, et al. Transdermal testosterone
pretreatment in poor responders undergoing ICSI: a randomized clinical trial. Hum Reprod
36. Fabregues F, Penarrubia J, Creus M, et al. Transdermal testosterone
may improve ovarian response to gonadotrophins in low-responder IVF patients: a randomized, clinical trial. Hum Reprod
37. Gougeon A. Dynamics of follicular growth in the human: a model from preliminary results. Hum Reprod
38. Gougeon A. Regulation of ovarian follicular development in primates: facts and hypotheses. Endocr Rev
39. Teissier MP, Chable H, Paulhac S, Aubard Y. Comparison of follicle steroidogenesis from normal and polycystic ovaries in women undergoing IVF: relationship between steroid concentrations, follicle size, oocyte quality and fecundability. Hum Reprod
40. Becerra-Fernández A, Pérez-López G, Román MM, et al. Prevalencia de hiperandrogenismo y síndrome de ovario poliquístico en transexuales de mujer a hombre. Endocrinol Nutr
41. Caanen MR, Soleman RS, Kuijper EAM, et al. Antimüllerian hormone levels decrease in female-to-male transsexuals using testosterone
as cross-sex therapy. Fertil Steril
42▪. Polyzos NP, Davis SR, Drakopoulos P, et al. T-TRANSPORT Investigators Group. Testosterone
for poor ovarian responders: lessons from ovarian physiology. Reprod Sci
This position paper from the T-TRANSPORT Investigators Group shows that today, evidence from clinical studies is not enough to recommend androgens in the poor responders. Hopefully, the T-TRANSPORT trial will give conclusive evidence on whether transdermal testosterone gel can improve the reproductive outcomes in this population.
43. Kim C-H, Ahn J-W, Moon J-W, et al. Ovarian features after 2 weeks, 3 weeks and 4 weeks transdermal testosterone
gel treatment and their associated effect on IVF outcomes in poor responders. Dev Reprod
44. Fooladi E, Bell RJ, Jane F, et al. Testosterone
improves antidepressant-emergent loss of libido in women: findings from a randomized, double-blind, placebo-controlled trial. J Sex Med
45. Nathorst-Böös J, Jarkander-Rolff M, Carlström K, et al. Percutaneous administration of testosterone
gel in postmenopausal women--a pharmacological study. Gynecol Endocrinol
46. Singh AB, Lee ML, Sinha-Hikim I, et al. Pharmacokinetics of a testosterone
gel in healthy postmenopausal women. J Clin Endocrinol Metab