Modern trend.
B~ E. w....h,lI.Dtj.~~_
FERTILITY AND STERILITY
Copyright " 1984 The American Fertility Society
Clomiphene citrate: mechanism(s) and site(s) of action-a
hypothesis revisited *
Eli Y. Adashi, M.D.
Division of Reproductive Endocrinology, Departments of Obstetrics/Gynecology and
Physiology, University of Maryland School of Medicine, Baltimore, Maryland
It has now been 28 years since the synthesis
I
of clomiphene citrate (CC) by Dr. Frank P. Palo-
poli,l, 2 then a synthetic chemist with the Wil-
liam S. Merrell Company. However, despite in-
tense basic and clinical research and a cumula-
tive bibliography now approaching 8000 contri-
butions, the mechanism(s) and site(s) of action of
CC have only been partially clarified. This
knowledge gap is particularly striking in view of
the widespread and highly successful use of CC
worldwide. Indeed, the clinical utility of CC has
recently been further enhanced through its intro-
duction as a primary agent in the management of
luteal phase dysfunction 3 and its application in
the area of in vitro fertilization. 4
It is the intent of this presentation to critically
explore the current hypothesis underlying the
mechanism(s) and site(s) of action of CC and to
propose its expansion and modification in keeping
with recently accumulated experimental data.
THE ESTROGENIC AND ANTIESTROGENIC
PROPERTIES OF CLOMIPHENE CITRA1'E:
HUMAN AND NONHUMAN PRIMATE STUDIES
Clomiphene citrate is a triphenylethylene de-
rivative substituted with a chloride anion and an
aminoalkoxy (OCH 2-CH2-N[C 2H 5h) side chain
(Fig. 1). The dihydrogen citrate moiety (C 6H s0 7)
simply denotes the fact that Clomid (Merrell Dow
Pharmaceuticals, Inc., Cincinnati, OH), as it is
clinically used, represents the dihydrogen citrate
salt form of CC proper.
*Supported in part by the Frank C. Bressler Research Fund.
Vol. 42, No.3, September 1984
Vol. 42, No.3, September 1984
Printed in U.SA.
It has been known since 1937 that triphenyl-
ethylene and triphenylchloroethylene are estro-
gen agonists of low potency but of unusually long
duration of action in the castrate or immature
rodent. 5,6 These traits have subsequently been
shown to be augmented by the alkoxy substitu-
tion. 7 Paradoxically, however, these early obser-
vations received relatively little attention until
the "rediscovery" that CC may act as an estrogen,
rather than an antiestrogen, in several species,
including the human. S Thus, CC is best viewed as
a mixed (agonist/antagonist) estrogen receptor
probe.
In the human, acting as an antiestrogen, CC
has been shown to attenuate vaginal cornifica-
tion,9 antagonize several estrogen-dependent
properties of cervical mucus,10 and reverse be-
nign and malignant endometrial changes. n , 12
Treatment with CC has also been shown to an-
tagonize the effects of exogenous ethinyl estradiol
on vaginal cytology, cervical mucus, and uterine
histology of castrate women. 13 More recently,
Kokko et al. 14 reported that the administration of
CC to untreated or estrogen-treated postmeno-
pausal women resulted in progressive endometri-
al atrophy and a diminution of the endometrial
concentrations of cytosolic estrogen and proges-
terone receptors. At the level of the hypothalamic
pituitary unit, concomitant administration of CC
to normally cycling women abolished the nega-
tive feedback effect of ethinyl estradiol on pitu-
itary gonadotropin release. 15 In addition, CC is
well known for its ability to elicit hot flashes in
premenopausal women 16 and to diminish the ca-
pacity of estrogen to relieve this climacteric
symptom. 17 In contrast, several investigators IS-20
Adashi Hypothesis of clomiphene citrate 331

2- [p-(2-chloro-l, 2-diphenylvinyt) phenoxy] triethylamine dihydrogen citrate
Figure 1
CC: structural formula.
have demonstrated the ability of CC to act as an
estrogen by suppressing the release of pituitary
gonadotropins in postmenopausal women. More
recently, Natrajan and Greenblatt21 demonstrat-
ed the ability of zuclomiphene citrate (formerly
the cis isomer of CC 22 ) to advance vaginal cytol-
ogy in castrate women, although little or no en-
dometrial proliferation was noted.
In the nonhuman primate, Holtkamp23 as well
as Markus 24 have clearly shown the estrogenic
capacity of CC to advance vaginal cornification
and to produce estrogen-like changes in the "sex-
ual skin" of the castrate rhesus monkey. Signifi-
cantly, CC did not antagonize the effects of eth-
inyl estradiol on either of these two parameters.
These findings extend the earlier observations of
Robson,25 using triphenylethylene, the parent
molecule of CC. More recently, Clark et al. 26 dem-
onstrated the ability ofCC, at a supraclinical dose
level of 100 mg/day, to induce endometrial prolif-
eration in the ovariectomized baboon. In contrast,
Marut and Hodgen,27 using normally cycling fe-
male rhesus and cynomolgus monkeys, observed
CC-induced attenuation rather than augmenta-
tion of ovarian folliculogenesis. Specifically, CC
therapy at clinical dose levels was associated with
a marked decline of the circulating levels of 1713-
estradiol (E 2), despite concurrent elevation of the
circulating levels of pituitary gonadotropins, es-
pecially follicle-stimulating hormone (FSH). Sim-
ilarly, Westfahl and Resko 28 recently demon-
strated the ability of CC to antagonize the luteo-
lytic effect of E2 in the nonpregnant cynomolgus
macaque.
Taken together, the preceding observations
suggest that CC is capable of interacting with a
variety of estrogen-dependent tissues, including
the hypothalamic-pituitary unit, ovary, endome-
trium, cervical mucus-producing glands, and vag-
332 Adashi Hypothesis of clomiphene citrate
inal mucosa. It can be further concluded that in
both humans and nonhuman primates, the inter-
action of CC with the estrogen receptor displays
mixed agonistic/antagonistic properties. In this
review, the estrogenic and antiestrogenic proper-
ties of CC will be examined only in the context of
its ovulation-producing property. As will be dis-
cussed below, current evidence suggests that both
the estrogenic and antiestrogenic properties of
CC may participate in the initiation of ovulation.
IS CLOMIPHENE CITRATE A UNIQUE
TRIPHENYLETHYLENE DERIVATIVE?
Clomiphene citrate is but one member of a
large family of triphenylethylene derivatives
(Fig. 2). Some of these (e.g., nafoxidine or CI-628)
have not as yet found clinical utility but are wide-
ly used as estrogen receptor probes in the course
of basic investigation. In contrast, tamoxifen has
been proposed for use for a variety of oncologic
indications. Likewise, chlorotrianisene (Tace,
Merrell Dow Pharmaceuticals, Inc.) has been
widely applied for postpartum lactation suppres-
sion and estrogen replacement therapy. Signifi-
cantly, both tamoxifen 29 -32 and chlorotrianis-
ene33 , 34 have been studied for their ovulation-
producing capabilities and found to compare fa-
vorably with CC. These observations suggest that
the ovulation-producing capability ofCC may not
be unique and may be shared with other triphen-
ylethylene derivatives. It is tempting to speculate
that triphenylethylene derivatives heretofore un-
ITAMOXIFEN I
~ OCH'O
OCH,
o '"
CH10 CI I TRIPHENYLETHYLENE I
ICHLOROTRIANISENE I
~o
OCH"CH"NICH'I'
o '"
CH10
INAFOXIDINE I
Figure 2
Structural formula of representative triphenylethylene deri-
vatives.
Fertility and Sterility

PROGESTERONE <>-<>
14
DAY Of CYCl£
2B
Figure 3
CC-initiated ovulation: the neuroendocrine sequence.
tested may yield "new" agents with ovulation-in-
ducing properties comparable, if not superior, to
those ofCC. Needless to say, introduction of such-
new agents would require prolonged and exten-
sive testing, an unlikely prospect in the current
research and development scene.
CLOMIPHENE CITRATE-INITIATED
OVULATION: THE NEUROENDOCRINE
SEQUENCE
Shown in Figure 3 is the sequence of endocrine
events taking place in the course of a hypothetical
CC treatment cycle. The bar spanning days 1 to 5
of the cycle represents the period of administra-
tion ofCC. The daily circulating levels ofluteiniz-
ing hormone (LH), FSH, E 2 , and progesterone are
expressed in relative terms and in the absence of
unitage for the sake of generalization. Neverthe-
less, the data closely reflect in both substance and
spirit the experience of Ross et al. 35
It is generally accepted that the first endocrine
event attributable to the administration of CC is
the concomitant increase in the release of pitu-
itary gonadotropins. Following the discontinua-
tion of CC or shortly thereafter, both gonadotro-
pins gradually decline to a preovulatory nadir,
only to surge again at midcycle. Concomitant
with and presumably secondary to the rise in pi-
tuitary gonadotropins, a progressive and time-de-
pendent increase in the circulating levels of E2 is
observed, culminating in a preovulatory peak. As
Vol. 42, No.3, September 1984
shown in the lower panel of Figure 3, the circulat-
ing levels of progesterone increase in the event of
a "successful" ovulatory cycle, suggesting the
formation of a functional corpus luteum.
The sequence of events described above is best
referred to as "CC-initiated ovulation," as op-
posed to "CC-induced ovulation." This distinction
is not merely one of semantics. Rather, such ter-
minology denotes the temporal and causal rela-
tionship between the ovulation-producing agent
and the actual event of ovulation. Whereas the
administration of human chorionic gonadotropin
is temporally as well as causally related to the
ensuing (within 36 hours) rupture of the domi-
nant follicle, CC therapy may be initiated as ear-
lyas 14 days prior to the actual event offollicular
rupture. Thus, rather than "inducing" ovulation,
CC appears to set in motion ("initiate") a series of
endocrine events culminating in the generation of
a preovulatory gonadotropin surge and hence fol-
licular rupture. Consequently, it is proposed that
the mechanisms whereby CC brings about ovula-
tion be sought within the time frame ofits admin-
istration.
Although the circulatory fate of CC following
discontinuation of therapy remains uncertain,
calculations based on half-life estimates (- 5
days36) suggest finite residual levels up to and
possibly beyond the time of midcycle. Pending
further investigation, the role, if any, of such re-
sidual CC in the ovulatory process remains un-
known.
CLOMIPHENE CITRATE-INITIATED
OVULATION: CURRENT HYPOTHESIS
Current concepts favor the notion that the abil-
ity of CC to initiate an ovulatory sequence is due
primarily, and perhaps exclusively, to its ability
to be recognized by and interact with estrogen re-
ceptors at the level of the hypothalamus (Fig. 4).
The exact hypothalamic nucleus involved has not
been determined, although the arcuate localiza-
tion of the gonadotropin-releasing hormone
(GnRH) pulse generator37 argues for an arcuate
site of interaction. However, the inability to dem-
onstrate estrogen receptors within GnRH neu-
rons 38 suggests non-GnRH, perhaps catechol-
aminergic, neurons as the target cell. Acting in
its capacity as an antiestrogen, CC is thought to
displace endogenous estrogen from hypothalamic
estrogen receptor sites. In functional terms, this
has been interpreted to result in the alleviation of
Adashi Hypothesis of clomiphene citrate 333
 ~
~ ....~~I HYPOTHALAMUS
I
~o
OCH'_CH'_N.CC'H~ PIT:;:RY
·C,H,o, LH/FSH
0'" ~+I EBTRADIOL-171'
Figure 4
CC-initiated ovulation: current hypothesis.
the negative feedback effect exerted by endoge-
nous estrogens, presumably resulting in a favor-
able alteration in the characteristics of pulsatile
GnRH release. In any event, the resultant pat-
tern of GnRH release is though to bring about
"normalization" of the release of pituitary gonad-
otropins, followed in turn by recruitment, selec-
tion, dominance, and ultimately rupture of the
"chosen" ovarian follicle.
Clearly, the above scheme ascribes a primary
and essentially exclusive role to the interaction of
CC at the level of the hypothalamus, relegating
other components of the reproductive axis to a
secondary position. Consequently, events occur-
ring at either the pituitary or ovarian level repre-
sent secondary phenomena contingent upon the
primary interaction of CC at the hypothalamic
level.
In the following paragraphs we will discuss al-
ternative and perhaps complementary mecha-
nisms of action of CC. Special consideration will
be given to the possibility that the pituitary and
the ovary may not merely represent passive by-
standers in the endocrine cascade initiated by CC,
but that either organ, in its own right, may con-
stitute a legitimate and active participant in the
process.
DOES CLOMIPHENE CITRATE EXERT A
DIRECT HYPOTHALAMIC EFFECT?
Despite the prevalent notion that the hypo-
thalamus constitutes a critical site of interaction
of CC, direct evidence to support this contention
is sorely lacking. Indeed, definitive proof for the
current hypothesis would require direct mea-
surements of the circulating levels of GnRH in
the portal circulation so as to document a CC-in-
duced alteration in the pattern of GnRH release.
Although such measurements are not feasible in
the human, current technology allows the con-
334 Adashi Hypothesis of clomiphene citrate
I duct of such experiments in subhuman pri-
mates. 39 However, pending such experiments,
I support for a hypothalamic site of action for CC
derives from indirect in vivo and in vitro observa-
tions.
For CC to be able to interact at the level of the
hypothalamus, a primary requirement that needs
to be met is the existence of estrogen receptors.
That this indeed is the case has been amply doc-
umented in various species, including the rhesus
monkey.40, 41 In the hypothalamus, the binding is
densest in the peri ventricular area, the arcuate
nucleus being densely labeled throughout. More-
over, multiple studies in rodents have demon-
strated the ability of CC to bind to hypothalamic
estrogen receptors and to effect their transloca-
tion to the nucleus. 42 -45 Taken together, these
studies suggest that the requirement for an es-
trogen receptor is more than adequately satisfied
in the case of the hypothalamus.
Early evidence to support a hypothalamic site
of action of CC derives from multiple studies
wherein systemic administration 46-51 or hypotha-
lamic implantation52 , 53 of CC has been shown to
increase the hypothalamic content of bioactive
GnRH, gonadotropin release, and ovulation. More
recently, several investigators54 ,55 reported an
increase in the peripheral circulating levels of
immunoreactive GnRH in the course of CC ther-
apy. However, given the controversial nature of
peripheral radioimmunoassay measurements of
GnRH, the validity of these observations remains
uncertain.
A more contemporary approach was adopted by
Tobias et al.,56 whose findings demonstrate the
ability of CC to antagonize the estrogen-stimu-
lated increase of tyrosine hydroxylase activity of
tuberoinfundibular dopaminergic neurons of the
medial basal hypothalamus (MBH) of ovariecto-
mized female rats. Although the relevance of
these observations to the ovulation-producing
capability of CC in the human female is un-
known, the ability of CC to alter the economy of
MBH catecholamines strongly supports a hypo-
thalamic site of interaction. These findings also
suggest that the capacity of CC to alter the pat-
tern of GnRH release requires the intermediacy
of regulatory catecholaminergic systems.
Evidence for a hypothalamic site of action of CC
in women was recently provided by Liu et a1. 57 In
this study, gonadotropin pulse amplitude and fre-
quency were compared in control subjects and
CC-treated normally cycling women. The results
Fertility and Sterility
 u--->I HYPOTH~LAMU·I
OCH.-CH.-NIC.H.I. GnRH
beneficial effect of estrogens on the occurrence of
hot flashes in postmenopausal womenP (5) CC-
mediated antagonism of the negative feedback
_~~I PITUtTARY
effect of ethinyl estradiol (a hypothalamic as well
as pituitary phenomenon by some accounts) in

->17
premenopausal women. 15 (6) Relative inefficiency
of CC in initiating ovulation under estrogen-poor
(hypogonadal) conditions.

E.TRADlOL-17~

Figure 5
ee-initiated ovulation: does ee exert a direct pituitary effect?

DOES CLOMIPHENE CITRATE EXERT A

indicated a significant enhancement of gonado-
DIRECT PITUITARY EFFECT?
tropin pulse frequency but not amplitude by CC.
Since the pulsatile frequency of gonadotropin re- Although the interaction of CC with the pitu-
lease is governed by hypothalamic mechanisms, itary has been studied by several investigators,
these findings suggest that the ovulation-initia- these observations received relatively little at-
tion property of CC is exerted, at least in part, at tention, and their pertinence to the mechanism of
the level of the hypothalamus. action of CC is not widely appreciated (Fig. 5).
Perhaps the most elegant evidence in support of In order to study the ability of CC to interact
a hypothalamic site of action of CC was recently with pituitary estrogen receptors, the crude nu-
provided by Miyake et a1. 58 In this study, the clear fraction obtained from anterior pituitaries
effects of CC on GnRH and LH release were ex- of E 2 -treated rats was incubated with a saturat-
amined in a sequential, double-chamber superfu- ing concentration of 3H-E 2 in the presence or ab-
sion system using MBH and/or pituitary excised sence of increasing concentrations of E 2 , CC, or .
from normal female rats in diestrus. When the tamoxifen (Fig. 6).59 The relative affinity of CC
MBH and the pituitary were superfused in se- for nuclear pituitary estrogen receptors was lower
quence with 2 x 10 -10 M E 2 , a significant in- (33-fold) than that ofE 2 • These and previous stud-
crease in GnRH release was observed during and ies60-63 attest to the ability of CC to interact with
following the administration of 3 x 10- 8 M CC. pituitary estrogen receptors.
CC was without effect when administered in the A direct pituitary effect of CC is supported by
absence of E 2 • These findings clearly demonstrate four different experimental approaches.
the ability of CC to enhance GnRH release from
MBH exposed to the tonic influence of E 2 • Similar
ell
studies using subhuman primates have not been Z
performed. is 100 0-0 ESTRADIOL
Z
o-oCLOMID
Given the preceding observations, there re- i
...0 ~ _TAMOXIFEN
mains little doubt as to the involvement of the is 80
hypothalamus in CC-initiated ovulation. How- ca::-
ever, pending further studies, it is not possible to
......
.0
determine with certainty whether CC interacts at ....
ilia::
~Z
80

zo
the level of the hypothalamus in its capacity as an .:...u 40
estrogen or an anti estrogen. Nevertheless, the =at
...
111-

following observations favor an antiestrogenic U

;:)
20
role for CC at the level of the hypothalamus: (1) Z
u D i
CC-enhanced GnRH release from E 2 -exposed it
0
U
MBH.58 (2) CC-mediated antagonism of estrogen- III
L
10-· 10-" 10-7 10-" 10""
stimulated MBH tyrosine hydroxylase activity. 56 • COMPETING LIGAND 1M'
(3) CC-elicited hot flashes (a putative central ner- Figure 6
vous system phenomenon) in premenopausal Relative binding affinities of E 2 , ee, and tamoxifen to rat
women. 16 (4) CC-mediated antagonism of the pituitary estrogen receptors.

Vol. 42, No.3, September 1984 Adashi Hypothesis of clomiphene citrate 335

'"
,r--------A
WASH",'"
""--------1" )I
•
.TEST
L INTERVAL.,'
________
±ESTRADlOL-17P GnRH
±CLOMIPHENE CITRATE
Figure 7
Direct effect of treatment with CC on gonadotropin release by
cultured rat pituitary cells: experimental design.
Studies of the Effects of Treatment with CC on
Gonadotropin Release by Cultured Pituitary
Cells in Vitro
Collagenase-dispersed anterior pituitary cells
from adult, ovariectomized female rats were plat-
ed onto tissue culture dishes and pretreated for 48
hours in the presence or absence of E 2 , with or
without CC (Fig. 7).59 At the conclusion of this
pretreatment interval, the cells were thoroughly
washed and reincubated for an additional 5 hours
in the absence or presence of GnRH, after which
time the media were collected and assayed for
their FSH content by radioimmunoassay.
5h
Treatment of pituitary cells with E2 or CC re-
sulted in 4.3-fold and 3.3-fold increases in pitu- '
II
itary sensitivity to GnRH, respectively (Fig. 8).
These findings suggest that E 2 , as well as CC, is
J,
I I
capable of enhancing the GnRH-stimulated re-
lease of FSH. Similar observations were made
with respect to LH.
Because CC is usually given to estrogen-primed
subjects in vivo, the effect of concomitant treat-
ment with CC and E2 on the GnRH-stimulated
release of FSH was examined (Fig. 9).59 Com-
bined treatment of pituitary cells with E2 and CC
produced a 3.6-fold increase in pituitary sensitiv-
ity to GnRH over controls. These findings suggest
that, as in the case of LH,64 CC does not antago-
nize the sensitizing effect of E2 on the GnRH=
stimulated release of FSH, supporting the notion
that CC may act as an estrogen, rather than an
antiestrogen, at the pituitary level.
It should be noted, however, that subsequent
studies using cultured rat anterior pituitary cells
did not confirm the preceding observations. 65 Al-
though the reasons underlying this apparent dis-
crepancy remain uncertain, differences in exper-
imental design and conditions were implicated. In
contrast, studies using cultured ovine pituitary
cells have recently confirm~d that treatment with
zuclomiphene citrate (formerly the cis isomer of
CC) enhanced the GnRH-stimulated release of
LH.66

2.0
2.0
0-0 Control
___ E."".. (70" l1li1

I 1.'
<>--0 CIomId (70"'l1li1

z
S! 1.0 z
go o
t
1.0
:::)
iDo o
%
iDo
~ 0.& %
~ 0.5

i t
L~. -L..-""1~~c::-.-.J..--:1:-:!.J-,
01 ,.10.r
I
•
10-'
•
10-'
1""0-1:',.=--...1...-1""'0-:1:',==-.
12
o
GnRH 1M)
GnRH 1M)

Figure 8 Figure 9
Direct effect of treatment with CC by itself on gonadotropin Direct effect of treatment with CC and E2 on gonadotropin
release by cultured rat pituitary cells. release by cultured rat pituitary cells. .

336 Adashi Hypothesis of clomiphene citrate Fertility and Sterility

 t/--....,>! HYPOTH~LAMUSI
OCH.-CH.-NIC.H.I. 8nRH
o . ~,-_PI_Tu,;_A_R_Y~
CoH.o. LHlFSH
." _I L.....-----,Wi.----RY----,
ESTRADIOL-17~
Figure 10
CC-initiated ovulation: does CC exert a direct ovarian effect?
Studies of the Effects of CC on Pituitary
Gonadotropin Release in Centrally Lesioned
Rats
The possibility of a pituitary site of action for
ee received additional support from the elegant
in vivo studies of Docke. 67 This investigator was
able to induce corpus luteum formation by subcu-
taneous injections of ee in female rats rendered
anovulatory by electrolytic lesioning of the me-
dial preoptic-suprachiasmatic regions of the hy-
pothalamus. Similar observations were made in
female rats rendered anovulatory by postnatal
androgenization or continuous light exposure.
These findings suggest that ee is capable of ef-
fecting ovulation in vivo without the benefit of a
functional hypothalamic component.
Studies of the Effects of Treatment with CC on LH
Release by the Superfused Pituitary in Vitro
Using a sequential, double-chamber superfu-
sion system involving the MBH and/or pituitary
of normal female rats in diestrus, Miyake et al. 58
also addressed the possibility of a direct pituitary
effect of ee. Treatment with ee alone resulted in
a limited but distinct increase in pituitary LH
release. Significantly, however, this increase in
LH release was not accompanied by a concomi-
tant increment in hypothalamic GnRH release.
These findings give strong support to the possibil-
ity that ee, by itself, may exert a direct stimu-
latory effect on pituitary LH release without the
otherwise apparently obligatory involvement of
the hypothalamus.
Vol. 42, No.3, September 1984
Comparative Studies of the Interaction ofCC with
Hypothalamic and Pituitary Estrogen Receptors
Several investigators have noted that the pitu-
itary is more sensitive to ee than the hypothal-
amus. Specifically, Kato et al. 61 observed that
only V100 of the ee dose required for blocking the
uptake of 3H-E 2 into the hypothalamus would
provide adequate blockade in the anterior pitu-
itary. These results have recently been corrobo-
rated using considerably lower concentrations of
ee.68 Kurl and Morris63 have also observed de-
pletion of pituitary cytosolic estrogen receptors at
a lower dose than was required at the level of the
hypothalamus. In addition, Adashi et al. 69 re-
ported that the duration of alterations in receptor
distribution were substantially prolonged in the
pituitary, as compared with the hypothalamus.
These findings suggest dissimilar effects of ee in
the pituitary and hypothalamus, raising the in-
triguing possibility that the pituitary gland,
rather than the hypothalamus, is the major cen-
tral target site of ee.
Taken in aggregate, the preceding observations
strongly suggest that ee may exert a direct stim-
ulatory effect on gonadotropin release at the pitu-
itary level, independent of its ability to alter the .
pattern of GnRH release. In doing so, ee appears
to exert a direct estrogenic rather than an anties-
trogenic effect at the pituitary level, consisting in
part of sensitization of the gonadotroph to the
action of GnRH.
DOES CLOMIPHENE CITRATE EXERT A
DIRECT OVARIAN EFFECT?
The possibility of a direct effect of ee on ovar-
ian function has been the subject of interest for
over 20 years (Fig. 10). In fact, early clinical ob-
servations suggested that "the primary site of the
action of clomiphene is either directly upon the
enzyme systems involved in ovarian steroidogen-
esis or as a potentiator of gonadotropins in this
respect.,,70 These conclusions were based on the
observation that administration of ee was fol-
lowed by "an increase in estrogen output, early in
the course of therapy, that could not be correlated
with any preceding or accompanying increase in
urinary gonadotropins." According to this earlier
view, the ovary was the primary site of action of
ee, whereas the increase in the release of pitu-
itary gonadotropins represented a secondary phe-
nomenon.71 However, subsequent studies using
Adashi Hypothesis of clomiphene citrate 337
more sensitive radioimmunoassay techniques to
detect alterations in the serum, rather than the
urinary levels of gonadotropins, established that
the first endocrine event following the adminis-
tration of CC is an increase in pituitary gonado-
tropin release. 35 Indeed, it is the latter observa-
tions that formed the basis for the current hy-
pothesis concerning the mechanisms and sites of
action of CC.
Although there is little doubt that CC can and
probably does exert a direct effect on ovarian
function, the exact nature of the interaction ofCC
at the level of the ovary remains controversial. Of
the various ovarian compartments, the granulosa
cell compartment is clearly endowed with estro-
gen receptors 72 and hence is likely to constitute a
target site for CC. Given the dependence of the
oocyte on granulosa cell function, the germ cell
status could be modulated secondarily through
the intermediacy of the granulosa cell compart-
ment. Although CC has been reported to affect
oocyte function,73. 74 a detailed discussion of this
phenomenon exceeds the scope of this review. Re-
cent studies confirm the embryotoxic effect of CC
(using mouse embryos)75 as well as its ability to
advance rabbit oocyte maturation and degenera-
tion, an effect associated with retarded subse-
quent embryonic development. 76 It remains un-
certain whether the theca, an otherwise estab-
lished estrogen-target compartment, is affected in
any way by CC.
To systematically explore the effects of CC on
ovarian function, both inhibitory and stimulatory
effects will be considered.
CLOMIPHENE CITRATE-INDUCED INHIBITION OF
OVARIAN FUNCTION
In Vitro Studies: Effects of Treatment with CC on
Ovarian Steroidogenesis and Follicular Atresia
Using human corpus luteum slices, Hammer-
stein77 reported that exposure to CC inhibits the
basal but not the human chorionic gonadotropin-
stimulated incorporation of 14C-acetate into pro-
gesterone. Similar findings were recently report-
ed by Westfahl and Resko. 28 These observations
were subsequently extended by Laufer et al.,73
whose findings demonstrate the ability of CC to
inhibit both basal and LH-stimulated steroid ac-
cumulation by cultured rat preovulatory follicles.
Specifically, treatment with 2 mM of CC de-
creased progesterone accumulation by 97%, E2 by
338 Adashi Hypothesis of clomiphene citrate
90%, and testosterone by 65%. Follicles incubated
for 24. hours with CC exhibited dose-dependent,
atretic-like changes. More recently, Sgarlata et
al. 78 reported the ability ofCC to inhibit LH-stim-
ulated progesterone biosynthesis by cultured avi-
an granulosa cells with a median effective dose of
3 x 10 - 6 M and a maximalinhibitory dose of 5 x
10 - 5 M. This effect of CC was shown to be exerted
at the site distal to the generation of cyclic adeno-
sine 3':5' monophosphate, involving, in alllikeli-
hood, inhibition of cholesterol side chain cleavage
activity.
Although the preceding studies are of potential
interest, it should be pointed out that in neither
case was an effect observed at a concentration
lower than 10- 6 M. Although the circulating con-
centrations of CC in the human have not been
thoroughly evaluated, the information available
suggests levels in the range of 10- 7 M.79 Given
that the in vitro effects observed required concen-
trations two orders of magnitude in excess of
those observed in vivo, the validity of these ob-
servations remains uncertain.
In Vivo Studies: Effects of Treatment with CC on
the Ovarian Augmentation Reaction and
Ovarian Steroidogenesis
U sing immature, hypophysectomized female
rats, Harman et al. 80 demonstrated the ability of
cis CC to inhibit the ovarian weight gain response
to human chorionic gonadotropin and FSH. Simi-
lar results were recently reported by Nakano et
al. 81 Unfortunately, the doses of CC used (10 or
20 mg/kg body weight/day) are approximately
two to four times higher than the highest dose of
CC « 5 mg/kg body weight/day) employed in
women. More recently, however, Marut and Hod-
gen,27 using normally cycling rhesus and cyno-
molgus monkeys, employed a 5-day CC regimen
at a level equivalent (on a body weight basis) to
200 to 250 mg daily in women. Using this regi-
men, these investigators observed attenuation of
ovarian folliculogenesis, rather than augmenta-
tion of gonadotropin-dependent preovulatory
events. Specifically, ovulation was delayed in 18
of 18 treatment cycles whether therapy was ap-
plied in the early, mid, or. late follicular phase.
Significantly, CC treatment cycles were charac-
terized by a marked decline in serum E2 levels
during the 5 days of therapy, despite concurrent
elevation of the circulating levels of pituitary go-
nadotropins, especially FSH. These data were
Fertility and Sterility
 Stahler et a1. 84 and Sturm and co-workers85 dem-
.
48h
.
5h
onstrated that the application ofCC (5 x 10- 7 M
to 5 X 10- 6 M) resulted in a substantial (up to
2.5-fold) increase in the incorporation of labeled
precursors into the appropriate C I8 estrogenic
products. These findings extended earlier obser-
vations wherein CC was shown to stimulate hu-
man placental aromatase activity in vitro, albeit
at concentrations in excess of 10- 4 M.86. 87
±FSH ANDROSTENEDIONE
More recently, Zhuang et a1. 88 reinvestigated
±ESTROGEN
±CLOMIPHENE CITRATE
the direct effects oftreatment with CC on granu-
losa cell aromatase activity. Obtained from im-
Figure 11 mature, hypophysectomized, diethylstilbestrol-
Direct effect of treatment with CC on cultured rat granulosa treated rats, granulosa cells were initially main-
cell aromatase activity: experimental design.
tained in an androstenedione-free, serum-free
medium for a period of 48 hours (Fig. 11). During
taken to mean that a direct anti estrogenic effect this period of time, referred to as the induction
of CC imparted ovarian refractoriness to the pre- interval, granulosa cells were treated in the ab-
vailing, even enhanced, gonadotropin levels. sence or presence of FSH (to induce aromatase
These observations provide the strongest evi- activity), with or without CC or E 2. At the conclu-
dence thus far in support of a direct inhibitory sion of this induction interval, the media were
effect of CC at the level of the primate ovary. collected, and the cells were thoroughly washed
and reincubated for an additional 5 hours in an
CLOMIPHENE CITRATE-INDUCED STIMULATION OF androstenedione-supplemented, serum-free me-
OVARIAN FUNCTION dium. The media were then collected and assayed
for their estrogen content by radioimmunoassay.
In Vivo Studies: Effects of Treatment with CC on The accumulation of estrogen during the test in-
Ovarian Aromatase Activity terval was taken to reflect the level of aromatase
In an isolated study by Smith,82 it was shown activity acquired during the induction interval.
that the preoperative administration of CC to an The basal accumulation of estrogen was negli-
anovulatory woman produced an increase in the gible and was unaffected by treatment with CC
in vitro rate of incorporation of labeled acetate
into E2 by the excised ovary. This CC-induced :=
III
increase in ovarian estrogen biosynthesis was I:
subsequently confirmed by Engels et a1.,83 using
in vivo canine ovarian preparations. Specifically,
1
II. 80
aI
infusion of CC via the left ovarian artery pro- E
"iii
duced a significant increase in ovarian E2 produc- .5
tion as detected in the venous effluent of the ova- z 60
0
ry under study. Using concomitantly adminis- i=
c
..J
tered I4C-androstenedione, Engels et a1. 83 were ~
~ 40
also able to show that treatment with CC in- ~ Ii
u
creased the rate of conversion of I4C-androstene-
dione to I4C-E2 and I4C-estrone. The effects of CC
u
c
Z 20
)
in this setting were accomplished at concentra-
tions in excess of 10 - 4 M, concentrations unlikely
III
c.:I
0
/6
to be encountered in vivo.
II:
.... -~
en 0
III
C ~ FSH 10-'0 10-8 10-8 10-7
In Vitro Studies: Effect of Treatment with CC on o~ CLOMIDIMI
+
C/o.; FSH
Ovarian Aromatase Activity
Figure 12
Using a closed in vitro perfusion system of hu- Direct effect of treatment with CC by itself on cultured rat
man ovaries obtained during the follicular phase, granulosa cell aromatase activity.

Vol. 42, No.3, September 1984 Adashi Hypothesis of clomiphene citrate 339

&0
o C E2 CLO""iID FS.j"-FSHFSt't-FSt't-
+ + + suggesting an inhibitory effect for CC at the ovar-
E2 CLOMID E2
+ ian level. Consequently, pending further investi-
CLOMID
Figure 13
Direct effect of treatment with CC and E2 on cultured rat
granulosa cell aromatase activity_
by itself (Fig. 12). In contrast, treatment with
FSH resulted in a substantial increase in the ac-
cumulation of estrogen (aromatase activity).
However, concomitant treatment of granulosa
cells with increasing molar concentrations (10 -10
to 10- 7 M) of CC resulted in progressive and
dose-dependent increments in the FSH-stimulat-
ed estrogen accumulation with an apparent medi-
an-effective dose of 5 x 10- 9 M. These findings
suggest that CC is capable of synergism with FSH
in the induction of granulosa cell aromatase ac-
tivity.
Because CC is usually given to estrogen-primed
subjects in vivo, Zhuang et al. 88 examined the
effect of concomitant treatment with CC and E2
on FSH-stimulated granulosa cell aromatase ac-
tivity. Both E 289 and CC underwent synergism
with FSH in the induction of granulosa cell aro-
matase activity (Fig. 13). Significantly, CC was
unable to antagonize the E2 effect. These findings
suggest that CC may exert a direct estrogenic,
rather than an anti estrogenic, effect at the ovar-
ian level by sensitizing the granulosa cell to the
action of pituitary gonadotropins. Although no
complementary in vivo studies have yet been per-
formed with clinical doses of CC, recent studies90
using tamoxifen revealed enhancement of ovar-
ian estrogen biosynthesis in the absence of no-
ticeable alteration in the circulating levels of
FSH and LH throughout the follicular phase.
These results suggest that the ability of tamoxi-
340 Adashi Hypothesis of clomiphene citrate
fen to enhance ovarian aromatase activity may
involve a direct ovarian effect, possibly synergism
with unaltered levels of pituitary gonadotropins.
On balance, discounting for the moment studies
conducted with inordinately high concentrations
of CC, the preceding observations would seem to
favor a direct stimulatory effect of CC on ovarian
aromatase activity. Specifically, the studies of
Smith,82 Stahler et al.,84 Sturm et al.,85 and
Zhuang et al. 88 suggest that CC may either stim-
ulate ovarian aromatase activity in its own right
or else undergo synergism with pituitary gonado-
tropins in this regard. These conclusions, how-
ever, cannot, at the present time, be reconciled
with the convincing work of Marut and Hodgen27
gation, the exact nature of the interaction of CC
at the ovarian level remains under scrutiny.
CLOMIPHENE CITRATE-INITIATED
OVULATION: PROPOSED HYPOTHESIS
Based on the foregoing, it would appear that
the formulation of a revised hypothesis, comple-
mentary to the one existing, may be in order (Fig.
14). Accordingly, it is proposed that the overall
profertility effect of CC on the reproductive axis
may not only reflect the consequences of its interac-
tion with the hypothalamus but, rather, may rep-
resent the sum of its direct effects at the hypotha-
lamic, pituitary, and ovarian levels. Given that
CC is an estrogen receptor probe and that all
levels of the reproductive axis are generously en-
dowed with estrogen receptors, it follows, by defi-
nition, that CC will be recognized by and interact
with every estrogen receptor-containing tissue.
That this indeed may be the case was convincing-
ly brought to light by the observations of Schultz
et al.,91 whose findings indicate preferential ac-
cumulation of 14C_CC in estrogen-target tissues
....IIII_~1
~ I
.±
HYPOTHALAMUS
~-;~::. ~ I
~, ~I+
ESTRADIOL-17~
Figure 14
CC-initiated ovulation: proposed hypothesis.
Fertility and Sterility
such as the pituitary, hypothalamus, ovary, liver,
uterus, and adrenal gland. Although the relative
significance of the contributions of the various
components of the reproductive axis remains un-
certain, it is hoped that future studies might clar-
ify this point. Whereas CC may act as an anties-
trogen at the level of the hypothalamus, most of
the data available suggest an estrogenic, rather
than an antiestrogenic, effect at the pituitary and
ovarian levels.
FUTURE DIRECTIONS: THE MANY FACES OF
CLOMIPHENE CITRATE
Earlier studies on the structure-activity rela-
tionships of muscarinic antagonists suggested
that CC might incorporate an acetylcholine-like
molecular arrangement. 92 Subsequent studies to
investigate possible interaction between CC and
muscarinic receptors demonstrated the ability of
CC to displace muscarinic antagonists from their
receptor-ligand complexes in various brain re-
gions, including the hypothalamus. 93 Given that
the binding affinity of CC to muscarinic receptors
is much higher than that of known muscarinic
antagonists, it is possible that CC might be acting
as a cholinergic agonist or partial agonist rather
than as an antagonist. However, in the guinea pig
ileum, CC antagonizes acetylcholine-mediated
contractions,92 suggesting an antagonistic func-
tion in these peripheral muscarinic receptors. Al-
though the preceding findings are of potential in-
terest, the possibility of nonspecific interaction
cannot, at present, be entirely ruled out. N ever-
theless, these findings suggest the possibility that
the ovulation-producing property of CC may be
exerted not only through specific estrogen recep-
tors but may also involve interaction with other
receptor systems thus far recognized. In this con-
text, it is of interest to note the long-standing
suggestion that CC may interact with histamine
receptors, given its ability to suppress the decid-
ual response (a putative histamine-dependent
process).94 Although this possibility has not been
further investigated, it should be pointed out that
the configuration of the aliphatic portion of the
CC molecule bears substantial similarity to sev-
eral histamine receptor probes.
Acknowledgments. The encouragement and support of Dr.
M. Carlyle Crenshaw and the invaluable assistance of Ms.
Donna Wirt are greatly appreciated.
Vol. 42, No.3, September 1984
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Received April 23, 1984.

Reprint requests: Eli Y. Adashi, M.D., Division of Reproductive Endocrinology, Departments of Obstetrics/Gynecology and
Physiology, University of Maryland School of Medicine, Room 11-009 Bressler Research Building, 655 West Baltimore Street,
Baltimore, Maryland 21201.

344 Adashi Hypothesis of clomiphene citrate Fertility and Sterility