REVIEW: Aromatase Inhibitors for Ovulation Induction

REVIEW: Aromatase Inhibitors for Ovulation Induction

Robert F. Casper and Mohamed F. M. Mitwally

Division of Reproductive Sciences (R.F.C.), Fran and Lawrence Bloomberg Department of Obstetrics and Gynecology,Samuel Lunenfeld Research Institute, Mount Sinai Hospital and The University of Toronto, Toronto, Ontario, Canada M5S2X9; and Division of Reproductive Endocrinology and Infertility (M.F.M.M.), Department of Obstetrics and Gynecology,Wayne State University, Detroit, Michigan 48202

Context: For the last 40 yr, the first line of treatment for anovulationin infertile women has been clomiphene citrate (CC). CC is a safe,effective oral agent but is known to have relatively common anties-trogenic endometrial and cervical mucous side effects that could pre-vent pregnancy in the face of successful ovulation. In addition, thereis a significant risk of multiple pregnancy with CC, compared withnatural cycles. Because of these problems, we proposed the concept ofaromatase inhibition as a new method of ovulation induction thatcould avoid many of the adverse effects of CC. The objective of thisreview was to describe the different physiological mechanisms ofaction for CC and aromatase inhibitors (AIs) and compare studies ofefficacy for both agents for ovulation induction.

Evidence Acquisition: We conducted a systematic review of all thepublished studies, both controlled and noncontrolled, comparing CCand AI treatment, either alone or in combination with gonadotropins,for ovulation induction or augmentation, identified through the En-trez-PubMed search engine.

Evidence Synthesis: Because of the recent acceptance of the conceptof using AIs for ovulation induction, few controlled studies were iden-tified, and the rest of the studies were pilot or preliminary compar-isons. Based on these studies, it appears that AIs are as effective asCC in inducing ovulation, are devoid of any antiestrogenic side effects,result in lower serum estrogen concentrations, and are associatedwith good pregnancy rates with a lower incidence of multiple preg-nancy than CC. When combined with gonadotropins for assisted re-productive technologies, AIs reduce the dose of FSH required foroptimal follicle recruitment and improve the response to FSH in poorresponders.

Conclusions: Preliminary evidence suggests that AIs may replaceCC in the future because of similar efficacy with a reduced side effectprofile. Although worldwide experience with AIs for ovulation induc-tion is increasing, at present, definitive studies in the form of ran-domized controlled trials comparing CC with AIs are lacking. (J ClinEndocrinol Metab 91: 760–771, 2006)

FOR THE LAST 40 yr, the first line of treatment for anovu-lation in infertile women has been clomiphene citrate

(CC). The choice of CC was appropriate because the drugwas highly effective in inducing ovulation in selected pa-tients with the advantages of being orally administered, rel-atively safe, and inexpensive. In contrast, alternative treat-ments usually involved parenteral gonadotropins that weresignificantly more complicated and uncomfortable to ad-minister, expensive, and associated with more frequent andserious complications. However, CC was also found to haveadverse effects, especially in the form of common antiestro-genic endometrial and cervical mucous changes that couldprevent pregnancy in the face of successfully induced ovu-lation. We believed it was time to develop a more effectiveovulation induction agent, and we have demonstrated thataromatase inhibitors (AIs) seem to be a promising alternativeto CC for ovulation induction. The purpose of this reviewwas to outline the data collected by us and others, over thepast 5 yr, regarding the use of AIs for ovulation induction.We will start with a brief description of the physiology of thenormal ovulatory cycle, information required for the reader

to understand the mechanism of action and problems withCC as well as the mechanism of action and advantages of AIs.

Physiological Basis of Ovulation

The ovarian cortex at puberty contains hundreds of thou-sands of primordial follicles (1). In response to unknownsignals, independent of gonadotropins, a cohort (hundreds)of primordial follicles is recruited to grow (2). During thisearly follicle development, the oocyte enlarges and the gran-ulosa cells proliferate to form a preantral follicle. Over 3–6months, the follicle develops FSH receptors in the granulosacells and LH receptors in the theca cells, and the follicle formsa fluid-filled space called an antrum (1). At this stage, antralfollicles become acutely dependent on FSH for further de-velopment (1). In natural cycles just before menses, fallingestrogen levels result in withdrawal of negative feedbackcentrally leading to increased gonadotropins levels (3). FSHstimulates granulosa cell proliferation and differentiation,with the development of more FSH receptors and the pro-duction of aromatase (4). LH stimulates androstenedioneproduction by theca cells that diffuses into the granulosa cellproviding substrate for estrogen secretion. This step is cat-alyzed by the aromatase enzyme, which is induced by FSH.The so-called two-cell, two-gonadotropin theory (5, 6) pos-tulated that FSH concentrations must exceed a certain level(FSH threshold) before follicular development will proceed.The duration of this period in which the threshold is ex-ceeded (the FSH window) is limited in the normal cycle bya gradual decrease in FSH, occurring in the early-midfol-

First Published Online December 29, 2005Abbreviations: AI, Aromatase inhibitor; CC, clomiphene citrate;

COH, controlled ovarian stimulation; E2, estradiol; ER, estrogen recep-tor; hCG, human chorionic gonadotropin; IUI, intrauterine insemina-tion; IVF, in vitro fertilization; OHSS, ovarian hyperstimulation syn-drome; PCOS, polycystic ovary syndrome.JCEM is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the en-docrine community.

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licular phase as a response to negative feedback from risingestrogen levels produced by the larger follicles (7). Smallerfollicles, with fewer FSH receptors, are no longer stimulatedto grow by FSH levels below the FSH threshold and undergoatresia (8). Therefore, generally only one follicle reaches thestage of ovulation each cycle, despite the fact that hundredsof primordial follicles, the number of which varies depend-ing on a woman’s age, may have begun development in thesame cohort 3–6 months earlier.

Administering exogenous FSH prolongs the time FSH lev-els are above the FSH threshold and extends the FSH win-dow. This allows multiple ovulation by rescuing smallerfollicles that would otherwise have undergone atresia. Inaddition, FSH also induces LH receptors in larger antralfollicles above 1.0 cm in diameter (9). At this point, LH cansubstitute for FSH in stimulating follicle growth and aro-matase activity (10). This observation led Sullivan et al. (11)and Filicori et al. (12) to postulate that exogenous LH/humanchorionic gonadotropin (hCG) could be used as a substitutefor FSH to continue follicular maturation before ovulation.

Rapidly increasing levels of estradiol produced by themature preovulatory follicle precede the midcycle LH andFSH surge that will initiate ovulation. The duration andcirculating level of estradiol seem to be the determinant of thetiming of the LH surge. Although the actual trigger of thesurge is unclear, there is the suggestion that it may be aresponse to a rise in both estrogen and progesterone (13),with a role for diurnal and estradiol-dependent changes inGnRH neuron firing activity (14). Angiogenesis and lutein-ization of granulosa and theca cells occur to form the corpusluteum in which an alteration of the steroidogenic pathwayresults in progesterone as the primary steroid hormone pro-duced after luteinization. The corpus luteum retains the abil-ity to produce estrogen. In addition, the demonstration ofmultiple waves of follicular growth in the majority of cycles(15) also suggests that some estrogen in the luteal phase maybe contributed by growing follicles that will subsequentlyundergo atresia. With implantation and pregnancy, hCGproduction by the trophoblast results in maintenance of cor-pus luteum function. Luteal progesterone is required untilabout 8 wk gestation when the placenta takes over proges-terone production (16). In the absence of pregnancy, LHlevels likely become too low to sustain the corpus luteum, asa result of very infrequent LH pulses (17). The corpus luteumundergoes regression with a fall in progesterone and estro-gen and the onset of menses. FSH levels rise with withdrawalof estrogen negative feedback and the next cohort of folliclesbegins to develop. The laboratory of Yen and colleagues (18)demonstrated, using naloxone (narcotic antagonist) infu-sions, that increased central endogenous opioid peptide ac-tivity is the cause of the decreased pulse frequency in theluteal phase. Such endogenous opioid activity is induced byelevated levels of progesterone.

In summary, normal follicular development culminates inovulation of a mature oocyte, followed by the developmentof a corpus luteum producing adequate amount of proges-terone. This sequence of events is orchestrated by the inter-action of local ovarian factors and endocrine factors from thepituitary and hypothalamus. The presence of subtle abnor-malities despite the occurrence of ovulation may be respon-

sible, at least in part, for unexplained infertility in somewomen, and perhaps for endometriosis-related infertility.On the other hand, overt anovulation or oligoovulation hasbeen traditionally classified into three groups: World HealthOrganization (WHO) types I, II, and III anovulation.

WHO Classification of Anovulation

Women in WHO group I suffer from a defect at the levelof the hypothalamus/pituitary. They are estrogen deficientwith normal or low FSH or prolactin levels. They typicallyhave amenorrhea and do not bleed in response to progestinchallenge. They generally require pulsatile GnRH infusionsor injectable gonadotropins to ovulate.

Women in WHO group II are not estrogen deficient. TheirFSH and prolactin levels are normal. They typically experi-ence oligomenorrhea, but they may have anovulatory cyclesor amenorrhea with bleeding in response to a progestin chal-lenge. This is the most common type of anovulation andincludes women with polycystic ovary syndrome (PCOS).For this group, oral agents such as insulin sensitizers, CC, ormore recently AIs are useful for ovulation induction.

Women in WHO group III include those with elevatedgonadotropins secondary to primary ovarian failure mainlydue to diminished ovarian reserve and loss of ovarian fol-licles. They are resistant to various methods of ovarian stim-ulation, and the best approach for their infertility is oocytedonation.

The present review will focus on the use of the oral agents,CC and AIs, to induce ovulation in women with WHO groupII anovulation.

Clomiphene Citrate

For more than 40 yr, CC has been the most commonly usedoral agent for induction of ovulation. In 1961 Greenblatt et al.(19) published the first results of ovulation inductionachieved by CC (at that time known as MRL/41). Since theearly 1960s, pregnancy rates with CC treatment have notchanged appreciably, despite advances in cycle monitoring.It is interesting to note that CC is considered pregnancy riskcategory X. This is particularly important when consideringits relatively long clearance half-life of about 5 d to 3 wk(depending on the isomer) and that CC may be stored inbody fat (20, 21).

Chemical structure and pharmacokinetics

Chemically, CC is a nonsteroidal triphenylethylene deriv-ative that exhibits both estrogen agonist and antagonist prop-erties, i.e. selective estrogen receptor modulating activity. CCis a racemic mixture of two distinct stereoisomers, enclomi-phene and zuclomiphene, having different properties. Avail-able evidence indicates that enclomiphene is the more potentantiestrogenic isomer primarily responsible for the ovula-tion-inducing actions of CC (20, 21). Currently available clo-miphene compounds are skewed toward enclomiphene pre-dominance. Levels of enclomiphene rise rapidly afteradministration and fall to undetectable concentrations aftera few days. Zuclomiphene is cleared far more slowly; levelsof this less active isomer remain detectable in the circulation

Casper and Mitwally • Aromatase Inhibitors for Ovulation Induction J Clin Endocrinol Metab, March 2006, 91(3):760–771 761



for more than 1 month after treatment and may actuallyaccumulate over consecutive cycles of treatment (21).

Mechanism of action of CC

CC binds to estrogen receptors (ERs) throughout thebody due to its structural similarity to estrogen. CC bind-ing to ERs occurs for an extended period of time, i.e. weeksrather than hours as with natural estrogen. Such extendedbinding ultimately depletes ER concentrations by inter-fering with the normal process of ER replenishment (19,22). The antiestrogenic effect on the hypothalamus, and thepituitary, is believed to be the main mechanism of actionfor ovarian stimulation. Depletion of hypothalamic ERsprevents correct interpretation of circulating estrogen lev-els; estrogen concentrations are falsely perceived as lowleading to reduced estrogen-negative feedback on GnRHproduction and subsequent increased gonadotropin (FSHand LH) secretion (Fig. 1). The rise of FSH promotesgrowth of ovarian follicles and ovulation in anovulatorywomen. It is believed that the hypothalamus is the mainsite of action because in normally ovulatory women, CCtreatment was found to increase GnRH pulse frequency(23). However, actions at the pituitary level may also beinvolved because CC treatment increased pulse amplitudebut not frequency in anovulatory women with PCOS, inwhom the GnRH pulse frequency is already abnormallyhigh (24).

During CC treatment, levels of both LH and FSH undergoa prolonged rise, compared with a natural cycle (Fig. 2A) (25)because of the prolonged ER depletion in the brain. Supra-physiological levels of estrogen can occur without centralsuppression of FSH because the normal ER-mediated feed-back mechanisms are blocked as a result of prolonged ERdepletion (Fig. 1). This pertubation of normal negative feed-back is reflected in the FSH window being extended, leadingto multiple follicle growth and a higher multiple pregnancyrate with CC than occurs in natural cycles.

Indications

The two main indications for CC treatment are inductionof ovulation in anovulatory infertility and stimulating mul-tifollicular ovulation or enhancing ovulation in ovulatoryinfertile women, e.g. unexplained infertility. For anovulatoryinfertility, CC treatment will likely be effective only in con-ditions in which adequate levels of circulating estrogen existto exert estrogen-negative feedback on gonadotropin pro-duction, which in turn could be antagonized by the anties-trogenic effect of CC as explained above. Adequate estrogenlevels for a response to CC are hard to determine in absolutevalues. However, a withdrawal bleed after progesterone ad-ministration is usually a reasonable clinical evidence for ad-equate levels of circulating estrogen. CC is the initial treat-ment of choice for most anovulatory or oligoovulatoryinfertile women who are euthyroid and euprolactinemic buthaving adequate circulating levels of estrogen (WHO type IIanovulation, e.g. PCOS. On the other hand, women with verylow circulating estrogen levels such as (WHO types I and III)or women with a defective hypothalamic/pituitary axis such

as Sheehan’s syndrome and Kallmann’s syndrome will notrespond to CC treatment.

For ovulatory infertility, CC is believed to enhance chancesof achieving pregnancy by stimulating multifollicular devel-opment as well as alleviating possible subtle ovulation dys-function (26).

Regimens of CC administration

CC is administered orally, usually starting on the secondto fifth day of spontaneous or progestin-induced menses.Treatment typically begins with a 50-mg tablet daily for5 consecutive days, increasing by 50-mg increments insubsequent cycles until ovulation is induced. After ovulationis achieved, the same dose is repeated until pregnancy isachieved or a maximum number of around six cycles isreached. Once the effective dose of CC is established, thereis no indication for further increases in dose unless the ovu-latory response is lost.

FIG. 1. CC (d 5). Administration of CC from d 3 to 7 results in ERdepletion at the level of the pituitary and mediobasal hypothalamus.As a result, estrogen-negative feedback centrally is interrupted andFSH secretion increases from the anterior pituitary leading to mul-tiple follicular growth. D 10, By the late follicular phase, because ofthe long tissue retention of CC, there continues to be ER depletioncentrally, and increased E2 secretion from the ovary is not capable ofnormal negative feedback on FSH. The result is multiple dominantfollicle growth and multiple ovulation. AI (d 5), Administration of anAI from d 3 to 7 results in suppression of ovarian E2 secretion andreduction in estrogen-negative feedback at the pituitary and medio-basal hypothalamus. Increased FSH secretion from the anterior pi-tuitary results in stimulation of multiple ovarian follicle growth. D 10,Later in the follicular phase, the effect of the AI is reduced and E2levels increase as a result of follicular growth. Because AIs do notaffect ERs centrally, the increased E2 levels result in normal negativefeedback on FSH secretion, and follicles less than dominant folliclesize undergo atresia, with resultant monofollicular ovulation in mostcases. [Reproduced with permission from R. F. Casper: Fertil Steril80:1335–1337, 2003 (106). © The American Society for ReproductiveMedicine.]

762 J Clin Endocrinol Metab, March 2006, 91(3):760–771 Casper and Mitwally • Aromatase Inhibitors for Ovulation Induction



Outcome of CC treatment

Classically, CC treatment has been reported to induceovulation in 60–80% of properly selected candidates. Morethan 70% of those who ovulate respond at the 50- or 100-mgdosage level. Cumulative conception rates up to 70% wereobserved after up to three successfully induced ovulatorycycles, (27–29). In another study, cumulative conception rateof 73% was achieved within nine CC-induced ovulatory cy-cles (28). Overall, cycle fecundity is approximately 15% inwomen who ovulate in response to treatment, with higher

chance of pregnancy in the first cycles (27). It is important torealize that these figures apply to young women in whomanovulation is the sole reason preventing them from con-ceiving. In the reality of daily clinical practice, such a groupof patients does not frequently exist, particularly in a sub-specialty referral infertility practice, in which much lowerpregnancy rates are observed with CC induction of ovula-tion. Age, presence of other infertility factors, treatment his-tory, and duration of infertility in addition to androgen levelsare important factors affecting treatment outcome (28). Am-enorrheic women are more likely to conceive than oligo-menorrheic women, probably because those who alreadyovulate, albeit inconsistently, are more likely to have othercoexisting infertility factors. Generally speaking, failure toconceive within six CC-induced ovulatory cycles should beregarded as a clear indication to expand the diagnostic eval-uation to exclude other factors or change the overall treat-ment strategy when evaluation is already complete (29).

Adverse antiestrogenic effects associated with CC treatment

Because of the relatively long half-life of CC isomers, CCmay exert unavoidable antiestrogenic effects on peripheralestrogen targets (endocervix and endometrium) that likelyexplain the absence of pregnancy despite ovulation observedin some CC-treated patients. Numerous studies in womenand in various model systems have described adverse effectson the quality or quantity of cervical mucus, and endometrialgrowth and maturation (30–35). It is generally believed thatthese effects are most apparent at higher doses or after longerdurations of treatment (31–33).

The endometrium is believed to be one of the most im-portant targets of the antiestrogenic effect of CC and mayexplain a large part of the lower pregnancy rate and thepossible higher miscarriage rate with CC. A reduction inendometrial thickness below the level thought to be neededto sustain implantation was found in up to 30% of womenreceiving CC (32). This observation has been confirmed byother studies (34, 35). It is important to mention that theabsolute value for endometrial thickness, below which preg-nancy seems unlikely, may be different from one center toanother. This is obviously due to the inter- and intraobserverdifferences in measuring the endometrial thickness. How-ever, in general, an endometrium that is thinner that 5 or 6mm is usually associated with significant likelihood of failureto conceive (32, 34, 35). In addition, successful implantationrequires a receptive endometrium, with synchronous devel-opment of glands and stroma. Despite conflicting reports ofCC effects on the endometrium (32–38), possibly due to dif-ferent methodology used for endometrial assessment, a re-cent study prospectively applied a quantitative and objectivetechnique to study the effect of CC on the endometrium ina group of normal women. This study found CC to have adeleterious effect on the endometrium, demonstrated by areduction in glandular density and an increase in the numberof vacuolated cells (38). Decreased uterine blood flow duringthe early luteal phase and the periimplantation stage mayalso explain, at least in part, the poor outcome of CC treat-ment (39).

To summarize, the exact significance of adverse anti-

FIG. 2. A, Mean serum FSH levels in natural cycles (solid circles) andCC-treated cycles (open circles) in 10 normal young women. Data arenormalized to the day of the LH surge. Prolonged elevation if serumFSH is seen during CC treatment consistent with absent estrogen-negative feedback because of prolonged ER depletion. B, Mean serumFSH levels in natural cycles (solid circles) and letrozole-treated cycles(open triangles) in nine normal young women. Data are normalized tothe day of the LH surge. Serum FSH declines normally during theletrozole treatment consistent with an intact central feedback mech-anism, in which estrogen suppresses FSH secretion and results inmonofollicular ovulation. [Reproduced with permission from S. A.Fisher et al.: Fertil Steril 78:280–285, 2002 (25). © The AmericanSociety for Reproductive Medicine.]




estrogenic effects on pregnancy outcome in CC-treatedwomen is difficult to quantify. Individual variability exists,likely because of the complexity of ER replenishment andactivation and individual differences in the pharmacokinet-ics of CC. The discrepancy in the success rates in achievingpregnancy between women with unexplained infertility andwomen with PCOS in response to CC treatment (higher ratesin PCOS women) suggests that PCOS women may be lessvulnerable to the antiestrogenic effects of CC on peripheraltissues.

A New Method of Ovulation Induction

In our tertiary referral center for infertility, we frequentlyidentified women, especially with unexplained infertility,who had failed to conceive with up to a year of CC treatmentcycles before referral. Almost universally, we discovered anendometrial thickness less than 6 mm on transvaginal ultra-sound. As a result of the antiestrogenic effects of CC on theendometrium in these and other women, we attempted de-velopment of a novel oral ovulation method that would befree of these adverse side effects. Aromatase was identifiedas a potential target for suppressing estrogen-negative feed-back centrally and the availability of new specific AIs led toinitial studies with these drugs.

Aromatase InhibitorsChemical structure and pharmacokinetics

Aromatase is a microsomal cytochrome P450 hemopro-tein-containing enzyme (the product of the CYP19 gene) andcatalyzes the rate-limiting step in the production of estro-gens, i.e. the conversion of androstenedione and testosteroneto estrone and estradiol, respectively (40). Aromatase activityis present in many tissues, e.g. ovaries, brain, adipose tissue,muscle, liver, breast tissue, and malignant breast tumors. Themain sources of circulating estrogens are the ovaries in pre-menopausal women and adipose tissue in postmenopausalwomen (41).

Aromatase is a good target for selective inhibition becauseestrogen production is a terminal step in the biosyntheticsequence. A large number of AIs have been developed overthe last 30 yr with the most recent, third-generation AIslicensed mainly for breast cancer treatment in postmeno-pausal women. The third-generation AIs have been devel-oped over the last 10 yr after the clinical failure of the second-(Fadrozole and Formestane, developed around 15 yr ago)and first-generation (aminoglutethimide, developed morethan 30 yr ago) AIs. The clinical failure of the first twogenerations was mainly due to significant side effects asso-ciated with their use and the lack of satisfactory potency orspecificity in inhibiting the aromatase enzyme (42–44).

The third-generation AIs commercially available includetwo nonsteroidal preparations, anastrozole [ZN 1033 (Arimi-dex)] and letrozole [CGS 20267 (Femara)], and a steroidalagent, exemestane (Aromasin), and are available for clinicaluse in North America, Europe, and other parts of the worldfor treatment of postmenopausal breast cancer. Letrozoleand anastrozole are reversible, competitive AIs with consid-erably greater potency than aminoglutethimide (�1000times) and, at doses of 1–5 mg/d, reduce estrogen levels by

97% to more than 99% down to concentrations below detec-tion by most sensitive immunoassays. AIs are completelyabsorbed after oral administration with mean terminal half-life of approximately 45 h (range 30–60 h) with clearancemainly by the liver. Mild gastrointestinal disturbances ac-count for most of the adverse events, although these haveseldom limited therapy (45–47). Exemestane is a steroidal,suicide inhibitor of aromatase (aromatase inactivators) witha circulating half-life of approximately 9 h but potentially alonger effect to inhibit aromatase because it is irreversible(48).

Induction of ovulation with AIs

Mechanism of action of AIs. We postulated that it would bepossible to block estrogen-negative feedback, without de-pletion of ERs as occurs with CC, by administration of an AIin the early part of the menstrual cycle. Both circulatingestrogen (produced mainly by the ovarian follicles and pe-ripheral conversion of androgens in fat and other tissues) andlocally produced estrogen in the brain exert negative feed-back on gonadotropin release (49–52). Inhibition of aroma-tization will block estrogen production from all sources andrelease the hypothalamic/pituitary axis from estrogenic neg-ative feedback (Fig. 1). The resultant increase in gonadotro-pin secretion will stimulate growth of ovarian follicles. With-drawal of estrogen centrally also increases activins, whichare produced by a wide variety of tissues including thepituitary gland (53) and will stimulate synthesis of FSH bya direct action on the gonadotropes (54).

The selective nonsteroidal AIs have a relatively short half-life (�45 h), compared with CC, and would be ideal for thispurpose because they are eliminated from the body rapidly(55, 56). Because AIs do not deplete ERs, as does CC, normalcentral feedback mechanisms remain intact. As the dominantfollicle grows and estrogen levels rise, normal negative feed-back occurs centrally, resulting in suppression of FSH (Fig.2B) and atresia of the smaller growing follicles. A singledominant follicle, and monoovulation, should occur in mostcases (Fig. 1).

In women with PCOS, relative oversuppression of FSHmay be the result of excessive androgen produced from theovary being converted to estrogen by aromatization in thebrain. The AIs suppress estrogen production in both theovaries and brain. In the case of PCOS, therefore, AIs shouldresult in a robust increase in FSH release and subsequentfollicle stimulation and ovulation. The actual FSH release islikely to be blunted by the high levels of circulating inhibinfound in PCOS patients (57–60) that would not be altered byaromatase inhibition. In addition, as pointed out above, aro-matase inhibition does not antagonize ERs in the brain, andthe initiation of follicle growth accompanied by increasingconcentrations of both estradiol and inhibin results in a nor-mal negative feedback loop that limits FSH response, therebyavoiding the risk of high multiple ovulation and ovarianhyperstimulation syndrome (OHSS).

Peripheral mechanism of action. A second hypothesis that mayadd to the mechanism of action of the AIs in ovarian stim-ulation involves an increased follicular sensitivity to FSH.




This could result from temporary accumulation of intrao-varian androgens because conversion of androgen substrateto estrogen is blocked by aromatase inhibition. Recent datasupport a stimulatory role for androgens in early folliculargrowth in primates (61). Testosterone was found to augmentfollicular FSH receptor expression in primates, suggestingthat androgens promote follicular growth and estrogen bio-synthesis indirectly by amplifying FSH effects (62, 63). Also,androgen accumulation in the follicle stimulates IGF-I, whichmay synergize with FSH to promote folliculogenesis (64–67).It is likely that women with PCOS already have a relativearomatase deficiency in the ovary, leading to increased in-traovarian androgens (68, 69) that leads to the developmentof multiple small follicles responsible for the polycystic mor-phology of the ovaries. The androgens, as described above,may also increase FSH receptors making these PCOS ovariesexquisitely sensitive to an increase in FSH through eitherexogenous administration of gonadotropins (hence the highrisk of OHSS) or endogenous increases in FSH as a result ofdecreased central estrogen feedback induced by aromataseinhibition. In the latter case, a relatively small rise in FSH,because of a normal estrogen feedback loop as describedabove, generally leads to monofollicular ovulation, thusavoiding the occurrence of OHSS.

Another part of the peripheral hypothesis involves ERs inthe endometrium. It is possible that aromatase inhibition,with suppression of estrogen concentrations in the circula-tion and peripheral target tissues, results in up-regulation ofERs in the endometrium, leading to rapid endometrialgrowth once estrogen secretion is restored. Estrogen has beenshown to decrease the level of its own receptor by stimulatingubiquitination of ERs, resulting in rapid degradation of thereceptors. In the absence of estrogen, ubiquitination is de-creased allowing up-regulation of the ER and increasingsensitivity to subsequent estrogen administration (70). Thiscould increase endometrial sensitivity to estrogen resultingin more rapid proliferation of endometrial epithelium andstroma and improved blood flow to the uterus and endo-metrium (71). As a result, normal endometrial developmentand thickness should occur by the time of follicular matu-ration, even in the face of the observed lower-than-normalestradiol concentrations in AI-treated cycles.

Indications for AIs in induction of ovulation. As a result of themechanisms of action of the AIs described above, we pro-posed that AIs could be used alone for induction of ovulationor as an adjuvant in conjunction with exogenous FSH or othermedications to improve the outcome of ovulation induction.A major advantage of an AI used alone is the ability toachieve restoration of monofollicular ovulation in anovula-tory infertility, e.g. PCOS as a result of the intact estrogen-negative feedback loop as described above. An AI could alsobe used in conjunction with FSH injections to increase thenumber of preovulatory follicles that develop and improvethe outcome of treatment. An increase in intraovarian an-drogen concentrations during aromatase inhibition couldimprove ovarian response to exogenous gonadotropins byincreasing ovarian sensitivity to FSH (peripheral mechanismof action described above).

To summarize, the AI when used alone should result in a

predictable response with the development of one or twomature follicles and a significantly reduced risk for OHSSand multiple gestation. To achieve multiple ovulation, theaddition of FSH to the AI is likely necessary.

Clinical studies of AIs for ovulation induction

Induction of ovulation after CC failure. The first study we per-formed was a proof-of-concept trial in a group of 22 womenwho had failed to respond to CC and were about to move onto injectable gonadotropin treatment with its associated costsand risks (72, 73). We defined inadequate response to CC asfailed ovulation induction or ovulation with a very thin mid-cycle endometrium (�0.5 cm). Patients who failed to respondadequately to CC were offered the option of trying the AI,letrozole, as an alternative treatment for induction ofovulation.

In the first study group, 12 women with PCOS receivedletrozole 2.5 mg daily from d 3 to 7 after a progestin-with-drawal bleed. During letrozole treatment, ovulation oc-curred in nine patients (75%), including three of the fourpatients who were anovulatory with CC. The mean endo-metrial thickness in the women receiving letrozole for ovu-lation induction was 0.81 � 0.14 cm. Pregnancy was achievedin three cycles (25%), two of which were singleton clinicalpregnancies and one was a chemical pregnancy.

Patients in the second group were ovulatory women withunexplained or mild male factor infertility and endometrialthickness 0.5 cm or less on CC. Each patient subsequentlyreceived one letrozole treatment cycle of 2.5 mg from cycled 3 to 7. During letrozole treatment, all women ovulated withendometrial thickness greater than 7 mm. A singleton clinicalpregnancy (10%) resulted from timed intrauterine insemi-nation (IUI) in one unexplained infertility couple who de-veloped two follicles greater than 1.5 cm.

A prospective, randomized, controlled trial of CC vs. an AIfor augmentation of ovulation in women with unexplainedinfertility was carried out at about the same time by Biljanand colleagues (74) in Montreal. Twenty-four patients wererandomized to receive CC 100 mg daily from cycle d 3 to 7,and 26 patients received letrozole 2.5 mg daily from d 3 to7. Using standard hormonal and ultrasound monitoring, theinvestigators showed that the CC group had a mean of twopreovulatory follicles, whereas the letrozole group produceda mean of one. Estradiol levels on the day of hCG weresignificantly elevated in the CC group (2300 pmol/liter),compared with the letrozole group (600 pmol/liter). Endo-metrial thickness and blood flow were significantly less withCC (6.9 mm and pulsatility index of 3.6), compared withletrozole (8.6 mm and pulsatility index of 3.1) on the day ofhCG. The pregnancy rate was 5.6% in the CC group vs. 16.7%in the letrozole group (nonsignificant).

We concluded from these preliminary studies that AIswere an effective alternative to CC, particularly in cases withrecurrent CC failure. However, it is important to point outthat in cases of CC resistance (failure to ovulate) due to severeinsulin resistance or the use of CC for inappropriate indica-tions (e.g. hypothalamic amenorrhea or ovarian failure), theuse of an AI is also unlikely to be successful. The correctionof insulin resistance with an insulin sensitizer is the logical




approach in patients with insulin resistance. Alternativetreatments should be considered for other problems such asexogenous gonadotropin injection in patients with hypogo-nadotropic hypogonadism and oocyte donation for caseswith ovarian failure.

Since this first study, evidence supporting the success ofAIs in ovulation induction for infertility treatment has beenaccumulating (74–78). Most of the studies used the AI, letro-zole. However, anastrozole, another third-generation AI sim-ilar to letrozole, was used in other studies (78, 79). It iscurrently not known whether there are any clinically signif-icant pharmacological differences between letrozole andanastrozole (80), especially regarding efficacy of ovulationinduction (81).

AIs plus gonadotropins

Reducing FSH dose required for optimum controlled ovarian hy-perstimulation. We investigated the idea of combining an AIwith FSH injection to reduce the dose of FSH required toachieve optimum controlled ovarian stimulation (COH),without adverse antiestrogenic effects. We (82–84) and oth-ers (85, 86) found a significant reduction in the FSH doserequired (from 45 to 55%).

We also compared the use of an AI in conjunction FSHwith the use of CC in conjunction with FSH or FSH alone (83).The study included women with unexplained infertility andmild male factor infertility. Thirty-six women received an AIwith FSH in 42 treatment cycles, 18 women received CC withFSH in 19 cycles, and 56 women received FSH only in 91cycles. In this study, we found that cotreatment with an AIsignificantly reduced the FSH dose required during COH tothe same extent as did CC. The AI was not associated withantiestrogenic effects as demonstrated by the significantlylower endometrial thickness noted with CC treatment de-spite the significantly higher estradiol (E2) level. In addition,the pregnancy rate with an AI and FSH was equivalent toFSH alone and almost twice the level seen with CC and FSHtreatment (83).

It is known that the cost of FSH injections constitutes asignificant part of the expense of infertility treatment, espe-cially during assisted reproduction. We believe that the AIswill markedly reduce the cost of infertility treatment bydecreasing the FSH dose required for optimum ovarian stim-ulation. This could make assisted reproductive technologyavailable to a larger group of infertile couples.

Improving ovarian response to FSH stimulation in poor respond-ers. The reduction in the dose of FSH required observed inconjunction with an AI encouraged us to explore the use ofan AI to improve response to ovarian stimulation with FSHin poor responders. In an observational cohort study (82), 12patients with unexplained infertility and a poor response toovarian stimulation with FSH in at least two cycles (total of25 cycles of FSH-only stimulation) were studied. Thesewomen underwent COH for IUI and poor response definedas less than three follicles greater than 1.8 cm in diameter onthe day of the LH surge or hCG administration. In the ex-perimental cycles, letrozole was given at a dose of 2.5 mgfrom d 3 to 7 after onset of menses, and FSH injection was

started on d 7 of the menstrual cycle at a dose of 50–225 IU/d,depending on the number of developing follicles seen onultrasound. The aim was to achieve three preovulatory fol-licles on the day of hCG injection.

During FSH plus letrozole stimulation cycles, the meannumber of mature follicles was 3.3, which was significantlyhigher than in FSH-only cycles (1.9 follicles). The amount ofFSH required was significantly lower in the letrozole plusFSH cycles than the FSH-only cycles (616 � 454 vs. 1590 �708 IU, respectively). There was no significant differencebetween FSH only or letrozole plus FSH on the day of hCGadministration or endometrial thickness on the day of hCGadministration. Although there was no difference in the levelof E2 on the day of hCG administration, the amount ofestrogen per mature follicle was significantly lower withletrozole treatment, consistent with previous studies of usingan AI for ovulation induction. Three of the women conceiveda clinical pregnancy with the combined letrozole and FSHtreatment.

In this clinical trial, we demonstrated a benefit of aro-matase inhibition in improving ovarian response to FSHstimulation in poor responders. The improved response wasclearly shown by the significantly higher number of maturefollicles and significantly lower amount of FSH needed toachieve an adequate number of preovulatory follicles. Inaddition, the endometrium sustained implantation as dem-onstrated by three clinical pregnancies in the letrozole andFSH cycles.

Optimal dose of AIs for repeated administration

The optimal dose of each AI is not yet clear. In most of thestudies to date, the dose of letrozole (2.5 mg) or anastrozole(1.0 mg) typically used for breast cancer treatment in post-menopausal women has been chosen. Biljan et al. (85) in arandomized study comparing 2.5 and 5.0 mg of letrozole inwomen with unexplained infertility suggested that thehigher dose might be associated with more follicles devel-oping. However, the study was not large enough to dem-onstrate a significant advantage. A more recent study byHealey et al. (86) used a dose of 5.0 mg of letrozole togetherwith FSH in an overlapping regimen for superovulation inwomen undergoing IUI. Compared with FSH treatmentalone, the combination of letrozole and FSH resulted in areduction of gonadotropin dose, similar pregnancy rates, buta slight negative effect on endometrial thickness, likely be-cause the overlapping regimen advanced follicle growth andovulation and did not allow time for clearance of the largerdose of letrozole by the time of hCG administration. Simi-larly, a study using 7.5 mg letrozole from cycle d 3 to 7showed, for the first time, a thinning of the endometriumsimilar to CC (76). Based on current data, it is likely that theoptimal dose of letrozole for a 5-d course of treatment isbetween 2.5 and 5.0 mg, with higher doses resulting in per-sistence of aromatase inhibition and estrogen levels too lowfor normal endometrial development by the time of ovula-tion. For anastrozole, there are not yet enough data to de-termine the preferred dose, although it appears that the stan-dard 1-mg dose is too low for optimal follicle recruitment andovulation (78).




Adverse effects and concerns about using AIs for inductionof ovulation

Side effects of AIs. In clinical use, nonsteroidal AIs are gen-erally well tolerated. The main side effects are hot flushes,headaches, and leg cramps (87, 88). These adverse effectswere observed in older women with advanced breast cancerwho were given the AIs on a daily basis over several months.Fewer adverse effects would be expected in healthy youngwomen administered a short course of AI for induction ofovulation. In addition, our clinical experience with ovulationinduction has been fewer side effects such as hot flushes andpremenstrual syndrome-type symptoms with AIs, comparedwith CC.

Aromatase inhibition is associated with significantly lowerserum estrogen levels at midcycle and per mature folliclethan found with CC (73, 83). The question whether low orvery low intrafollicular estrogen is compatible with folliculardevelopment, ovulation, and corpus luteum formation hasbeen reviewed before (89). Markedly reduced to absent in-trafollicular concentrations of estrogen are known to be com-patible with follicular expansion, retrieval of fertilizable oo-cytes, and apparently normal embryo development (90–93).The rapid clearance of the AIs, the reversible nature of en-zyme inhibition, and elevated levels of FSH, which inducesnew expression of aromatase enzyme, are factors that limitaccumulation of androgens and likely result in increasingestrogen production that should be relatively normal at thetime of ovulation. This conclusion has now been confirmedby the use of AIs in in vitro fertilization (IVF) reviewed below.

Pregnancy outcome with AIs

We recently reported the clinical outcome of pregnanciesobtained through the use of AIs for ovulation induction orCOH for IUI (94). We described a cohort study comparing theoutcome of pregnancies achieved after letrozole and otherovarian stimulation treatments with a control group of preg-nancies spontaneously conceived without ovarian stimula-tion. In three tertiary referral centers over a 2-yr period, therewere 394 pregnancy cycles in 345 infertile couples (133 preg-nancies with 2.5 or 5 mg letrozole alone or gonadotropins,113 pregnancies with CC alone or with gonadotropins, 110pregnancies with gonadotropins alone, and 38 pregnanciesachieved without ovarian stimulation). Pregnancy rates inthe various treatment groups are shown in Fig. 3A. Preg-nancies conceived after AI treatment was associated withcomparable miscarriage and ectopic pregnancy rates, com-pared with all other groups including the spontaneous con-ceptions. In addition, letrozole use was associated with asignificantly lower rate of multiple gestation, compared withCC (4.3 vs. 22%, respectively) (Fig. 3B), consistent with ourhypothesis of an intact negative feedback loop centrally witharomatase inhibition.

Recent safety studies have found anastrozole to have noteratogenic or clastogenic effects in animal embryo devel-opment, whereas there have been some concerns regardingteratogenic effects of letrozole if administered unintention-ally during pregnancy (95). Because of the short half-life ofeach drug, administration in the early follicular phase shouldresult in clearance of the agents before implantation takes

place. Nevertheless, care should be taken in all cases of ovu-lation induction with these drugs to ensure that the patientis not pregnant before administration. We recommend that

FIG. 3. A, Pregnancy outcome in 3748 cycles of timed intercourse orIUI in women undergoing cycle monitoring with serial transvaginalultrasound scans and serum E2 and LH determinations. The columnlabeled Natural refers to women undergoing cycle monitoring andtimed intercourse or IUI with no drug treatment. B, Multiple gesta-tion rates associated with the different ovarian stimulation treat-ments. There were no multiple gestation cases among pregnanciesthat occurred spontaneously. Letrozole (Let) treatment, at both the2.5 and 5.0 mg/d doses, was associated with significantly lower mul-tiple gestation rates, compared with all other methods of ovarianstimulation. CC treatment (alone or with gonadotropins) was asso-ciated with significantly higher multiple gestation rates, comparedwith all other ovarian stimulation treatments. [Reproduced with per-mission from M. F. Mitwally et al.: Am J Obstet Gynecol 192:381–386,2005 (95). © Elsevier Inc.]




a blood �hCG level be ascertained before using AIs in pre-menopausal women.

Recently, Health Canada and Novartis Pharmaceuticalsissued a warning that letrozole should not be used for ovu-lation induction because of the potential for fetal toxicity andmalformations. This warning was based on an abstract byBiljan et al. (96) in which six congenital abnormalities and onecase of hepatocellular carcinoma were reported among 150births resulting from the use of letrozole for ovulation in-duction. These 150 babies were compared with a database ofover 36,000 spontaneously conceived babies born in a low-risk community hospital in Montreal. Although the overallanomaly rate was not increased, the authors reported a sig-nificant increase in locomotor and cardiac abnormalities inthe letrozole-treated pregnancies compared to the controls.We believe a larger group of letrozole babies needs to beassembled and compared to a more suitable control group ofinfertile women before any conclusion of an increase in con-genital anomalies with letrozole can be confirmed. We arenow in the process of collecting such data.

AIs for IVF

Despite recent developments in ovarian stimulation andassisted reproductive technologies, there has not been a cor-responding increase in implantation rates. Supraphysiologi-cal levels of estrogen may explain some of the adverse effectsof ovarian stimulation on infertility treatment, including del-eterious effects on the endometrium (97, 98) and embryo (99,100). Basir et al. (101) have shown that high response to COHfor IVF results in endometrial glandular and stromal dys-synchrony as assessed by morphometric analysis. A step-down protocol has been proposed for IVF to lower E2 con-centrations and improve successful implantation (102). Analternative approach is using an AI to significantly suppressE2 levels around d 3–7 of the menstrual cycle. After stoppingthe AI, E2 levels increase steadily to reach levels high enoughto trigger an LH surge around d 12–14 of the cycle. However,preovulatory levels of estrogen remained about half to onethird the levels seen in CC- or FSH-stimulated cycles (83).This strategy may work similarly to a step-down protocolwithout jeopardizing the potential number of oocytesretrieved.

Clinical studies of AIs for ovarian stimulation in IVF

In a randomized, controlled study, Goswami et al. (103)compared an AI plus FSH protocol with a standard GnRHagonist and FSH protocol in poor responders undergoingIVF. Although this was a small pilot study including only 38patients, the results suggested that the addition of an AI toa small dose of FSH (150 IU; two injections of 75 IU on cycled 3 and 8) resulted in a similar number of oocytes retrieved,embryos transferred, and pregnancy rate as observed in thewomen on the standard protocol receiving a mean (� sem)of 2865 � 228 IU FSH. The authors concluded that AIs couldbe a low-cost alternative to natural-cycle IVF in patients whoare poor responders to FSH and that larger randomizedstudies are required to confirm their data.

A recent, larger study by Garcia-Velasco et al. (104) eval-uated the use of an AI as an adjuvant to FSH treatment in IVF

cycles of poor-responder patients. In this study, 147 lowresponders, with at least one previous canceled IVF cycle,were enrolled. The study was prospective but not random-ized. The women were divided into a control group of 76patients treated with high-dose gonadotropins in a GnRH-antagonist regimen. The experimental group of 71 patientsreceived letrozole 2.5 mg plus gonadotropins for the first 5 dof stimulation followed by the same gonadotropin/antago-nist regimen.

The study demonstrated that patients receiving an AI hadhigher numbers of oocytes retrieved and had a higher im-plantation rate despite receiving the same doses of FSH/human menopausal gonadotropin as the control group. Bothtestosterone and androstenedione were significantly in-creased in concentration in the follicular fluid in the exper-imental group, compared with the controls. Interestingly,follicular fluid E2 levels were similar to controls. These find-ings are consistent with the hypothesis that aromatase inhi-bition, by blocking androgen to estrogen conversion, in-creases intraovarian androgens and follicular FSH receptorexpression and sensitivity to FSH administration and is rap-idly reversible.

Another reason to avoid elevated estrogen levels duringovarian stimulation is fertility treatment in women withbreast cancer. Treatment for this cancer often involves che-motherapy with alkylating agents that can damage ovarianfollicle reserve leading to premature ovarian failure. With therecent success of oocyte cryopreservation, some women areopting to freeze oocytes or embryos for later use by them-selves or a gestational carrier. Oktay et al. (105) studied 60women (age range 24–43 yr) with breast cancer. Twenty-ninewomen requested IVF before chemotherapy, and 31 womendid not undergo IVF and served as the control group. The 29patients underwent 33 ovarian stimulation cycles with eithertamoxifen 60 mg/d alone or in combination with low-doseFSH (TamFSH-IVF) or letrozole 5 mg in combination withFSH (letrozole-IVF). After IVF, all resultant embryos werecryopreserved. The study was not randomized for severalreasons. Compared with women receiving tamoxifen alone(1.5 mature oocytes and 1.3 embryos), there was a significantincrease in the mean number of mature oocytes retrieved inthe group receiving letrozole-IVF (8.5) and TamFSH-IVF(5.1). The mean number of embryos cryopreserved was alsosignificantly increased (5.3 and 3.8, respectively). Peak E2levels were significantly lower with letrozole-IVF, comparedwith TamFSH-IVF (380 � 57 and 1182 � 271 pg/ml, respec-tively). After almost 2 yr of follow-up, the cancer recurrencerate was similar between IVF and control patients. The au-thors concluded that both tamoxifen and letrozole added toFSH could increase the number of oocytes retrieved for IVFin breast cancer patients, but the letrozole protocol may bepreferred because it resulted in lower peak E2 levels.

Conclusion

Preliminary studies have demonstrated that AIs are effec-tive for ovulation induction in infertile women. Based on theevidence reviewed above, these oral agents seem to be effi-cient for ovulation induction. We believe that a major ad-vantage of AIs for ovulation induction is monofollicular ovu-




lation. Especially in PCOS patients, who are oftenhyperresponsive to gonadotropins, a drug that consistentlyresults in a single ovulation is very desirable. In addition,because negative effects peripherally on the endometriumare absent, a second advantage of AIs for ovulation inductionis minimal if any ultrasound monitoring is needed for en-dometrial thickness. Both of these advantages allow primarycare providers or community-based gynecologists, withoutready access to ultrasound monitoring, to participate in themanagement of PCOS and other anovulatory women.

In IUI cycles, AIs alone are probably not the optimal choicebecause, generally speaking, it is preferable to see two orthree mature follicles developing, depending on the patient’sage. To ensure multiple ovulation in IUI cycles, the additionof a low dose of FSH to the AI is required, although FSH canbe delayed (sequential protocol) until E2 levels begin to riseand when the exogenous FSH administration would over-ride negative feedback occurring centrally. This type of pro-tocol does not accelerate follicle development, and ovulationoccurs normally around cycle d 14. There is adequate time forendometrial growth to occur normally and a small dose ofgonadotropin is required, generally in the range of 50–75 IUdaily.

These advantages, if confirmed, suggest that AIs may bea viable option to replace CC in the future as the new primarytreatment for ovulation induction and in combination withFSH for assisted reproduction procedures. However, largerrandomized, controlled studies are required to determine thebest treatment regimen. If such large studies confirm effec-tiveness and safety, the use of AIs for ovulation inductionwould be the first improvement in oral ovulation inductionin decades.

Acknowledgments

Received August 29, 2005. Accepted November 29, 2005.Address all correspondence and requests for reprints to: Robert F.

Casper, M.D., F.R.C.S.(C), 150 Bloor Street West, Suite 210, Toronto,Ontario, Canada M5S 2X9. E-mail: [email protected].

This work was supported by the Canadian Institutes of Health Re-search, Ottawa, Ontario, Canada.

The authors have no conflict of interest.

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JCEM is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving theendocrine community.




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REVIEW: Aromatase Inhibitors for Ovulation Induction