REVIEW

Gonadotropins and Their Analogs: Current and

Potential Clinical Applications

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Ross C. Anderson,1,2 Claire L. Newton,1,3 Richard A. Anderson,4 and Robert P. Millar1,2,3,5,6

1
Centre for Neuroendocrinology, University of Pretoria, Pretoria 0001, South Africa; 2Department of
Physiology, Faculty of Health Sciences, University of Pretoria, Pretoria 0001, South Africa; 3Department of
Immunology, Faculty of Health Sciences, University of Pretoria, Pretoria 0001, South Africa; 4MRC Centre for
Reproductive Health, The Queen’s Medical Research Institute, University of Edinburgh, Edinburgh EH16 4TJ,
United Kingdom; 5Department of Integrative Biomedical Sciences, Faculty of Health Sciences, University of
Cape Town, Cape Town 7700, South Africa; and 6Institute of Infectious Diseases and Molecular Medicine,
University of Cape Town, Cape Town 7700, South Africa

ABSTRACT The gonadotropin receptors LH receptor and FSH receptor play a central role in governing reproductive competency/fertility.
Gonadotropin hormone analogs have been used clinically for decades in assisted reproductive therapies and in the treatment of various
infertility disorders. Though these treatments are effective, the clinical protocols demand multiple injections, and the hormone preparations
can lack uniformity and stability. The past two decades have seen a drive to develop chimeric and modified peptide analogs with more
desirable pharmacokinetic profiles, with some displaying clinical efficacy, such as corifollitropin alfa, which is now in clinical use. More
recently, low-molecular-weight, orally active molecules with activity at gonadotropin receptors have been developed. Some have excellent
characteristics in animals and in human studies but have not reached the market—largely as a result of acquisitions by large pharma.
Nonetheless, such molecules have the potential to mitigate risks currently associated with gonadotropin-based fertility treatments, such as
ovarian hyperstimulation syndrome and the demands of injection-based therapies. There is also scope for novel use beyond the current
remit of gonadotropin analogs in fertility treatments, including application as novel contraceptives; in the treatment of polycystic ovary
syndrome; in the restoration of function to inactivating mutations of gonadotropin receptors; in the treatment of ovarian and prostate
cancers; and in the prevention of bone loss and weight gain in postmenopausal women. Here we review the properties and clinical
application of current gonadotropin preparations and their analogs, as well as the development of novel orally active, small-molecule
nonpeptide analogs. (Endocrine Reviews 39: 911 – 937, 2018)

G lobally, many couples desiring children expe-

rience problems with infertility (). With the
global demand for assisted reproductive technologies
(ARTs), there has been a drive toward the discovery
and development of effective therapeutic modula-
tors of human reproduction. Central to this is the use
of gonadotropins [FSH, LH, and human chorionic
gonadotropin (hCG)] and their analogs. Here, we
Gonadotropins and Their Receptors
The hypothalamic-pituitary-gonadal axis
The hypothalamic-pituitary-gonadal (HPG) axis is an
endocrine signaling conduit that governs pubertal de-
velopment and reproductive competency in humans.
provide an up-to-date review of the development,
properties, and activities of peptide and nonpeptide
gonadotropin analogs and their current and potential
clinical uses. The authors searched PubMed and
Google Patents databases for original articles, appro-
priate reviews, and patent applications focused on
gonadotropin receptors, gonadotropins, gonadotropin
analogs, and ARTs published until February .
ISSN Print: 0163-769X
ISSN Online: 1945-7189
The core events that define this system include the Printed: in USA
pulsatile release of GnRH from ~ neurons in the Copyright © 2018
hypothalamus () into the hypophyseal portal capillary Endocrine Society
Received: 14 February 2018
blood from where it is delivered to the anterior pituitary.
Accepted: 25 June 2018
Binding and activation of its cognate receptor, the First Published Online:
GnRH receptor (GnRHR), on gonadotrope cells of the 2 July 2018

doi: 10.1210/er.2018-00052 https://academic.oup.com/edrv 911

REVIEW

ESSENTIAL POINTS
· Urinary gonadotropin analogs have been used clinically for decades in assisted reproductive therapies and in the
treatment of various infertility disorders
· Issues with the gonadotropin peptide hormones include clinical protocols requiring multiple injections, nonuniformity of
preparations, protein stability, and increased risk of ovarian hyperstimulation syndrome
· More recently, recombinant gonadotropin preparations have been favored as a result of their improved purity and batch-
to-batch consistency
· Gonadotropin hormones with differing half-lives and oral bioavailability may mitigate the need for repeated injections

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and reduce side-effects associated with current treatment regimens
· Several peptide analogs have been developed, one of which, an FSH analog with an increased half-life (corifollitropin alfa),
has entered the clinic
· A spectrum of orally available, low-molecular-weight gonadotropin analogs (agonists and antagonists) has been in
development, but none is yet commercially available, and many development programs have been terminated as a result
of pharmaceutical company mergers or acquisitions
· In addition to use in assisted reproductive therapies, gonadotropin analogs may have potential application as
contraceptives and in the treatment of polycystic ovary syndrome and ovarian and prostate cancers and prevention of
bone loss and weight gain in postmenopausal women

anterior pituitary stimulate the production and secre-
tion of the gonadotropin hormones LH and FSH, which
are released into the general circulation to stimulate
gonadal steroidogenesis, peptide hormone secretion
(inhibin and activin), and gametogenesis. Gonadal
steroids and peptide hormones then feed back in
negative and positive modalities at the hypothalamus
and/or pituitary (, ).
The actions of gonadotropins in the HPG axis
The gonadotropin glycoproteins exert their effects on
the HPG axis through interaction with and activation
of their cognate receptors, the LH receptor (LHCGR/
LHR) and FSH receptor (FSHR). Whereas a number of
studies have documented the presence of LHR and
FSHR in extragonadal tissues (, ), physiologically,
their primary site of activity is the gonads. FSHR
activation stimulates follicular development in females
and the initiation of spermatogenesis in males whereas
LHR stimulation is responsible for steroidogenesis and
follicular/gamete maturation, as well as the induction
of ovulation in females. LH regulates expression of
steroidogenic enzymes and the subsequent production
of androgens within the interstitial Leydig and thecal
cells of the testes and ovaries, respectively. Steroido-
genesis is also dependent on the activity of FSH in
testicular Sertoli cells and the granulosa cells of the
ovaries, supporting gonadal development and facili-
tating the conversion of androgen to estrogen via the
enzyme aromatase. Activation of FSHR additionally
results in the production of the TGF-b family
members activin and inhibin, which positively and
negatively feed back to the pituitary and hypothalamus
to regulate FSH production/activity ().
The gonadotropin-secretion profiles are also sex-
ually dimorphic, differing dramatically between males
and females. Whereas LH pulse frequency remains
constant in males, it varies over the female menstrual
cycle with low-frequency pulses during the luteal
phase of the cycle and higher-frequency pulses in the
follicular phase. FSH pulsatility is less discernible, as
unlike LH, FSH is not stored in secretion granules and
has a longer half-life in the circulation. In females, FSH
activity in the late-luteal/early-follicular phases of the
cycle is responsible for recruitment of five to seven
Graafian follicles into the menstrual cycle and drives
follicular development. Estradiol, produced by the
developing follicle, feeds back negatively to the hy-
pothalamus, resulting in suppressed LH secretion.
Through poorly defined mechanisms, an estradiol
threshold is reached, at which point estradiol switches
to positive feedback, resulting in a midcycle increase in
LH pulse frequency and surge of LH, which are re-
sponsible for initiating ovulation and luteinization of
the ruptured follicle. The luteinized follicle, or corpus
luteum, subsequently produces progesterone that
prepares the uterine endometrium for implantation.
Progesterone also feeds back negatively at the hypo-
thalamus, suppressing LH secretion, resulting in at-
rophy of the corpus luteum should implantation not
occur.
The importance of both glycoprotein hormones
and their receptors in gonadal function is highlighted
by the pathophysiologies attributed to inactivating (or
constitutively activating) mutations that affect the LH/
LHR and FSH/FSHR signaling modalities. Loss of
functional LH and FSH in males and females can
result in impaired fertility/infertility phenotypes and in
the case of LH, a lack of pubertal development (, ).
The severity of the phenotype attributed to muta-
tions in the LHR correlates with the degree of receptor
inactivation and a resultant spectrum of phenotypes from

912 Anderson et al Peptide and Nonpeptide Gonadotropin Analogs Endocrine Reviews, December 2018, 39(6):911–937

pseudohermaphroditism and hypogonadism through
to the less severe micropenis and oligomenorrhea ().
Interestingly, loss of FSH/FSHR signaling in females
results in infertility, in contrast to males, where
homozygous-inactivating FSH/FSHR mutations appear
to result in a qualitative and quantitative reduction in
spermatogenesis without rendering the patient com-
pletely infertile, implying a level of redundancy in FSHR
signaling with regard to male fertility (–). These
findings appear to be supported by an FSH-deficient
mouse model in which female mice had folliculogenesis
defects and were infertile, whereas males displayed im-
paired testes development but remained fertile ().
Regulation of gonadotropin secretion
In both male and female systems, the processes reg-
ulating the production and secretion of the gonado-
tropins are complex and represent an intricate
relationship among hormonal and steroidal produc-
tion, secretion, and feedback (, , ). As men-
tioned previously, the secretion of LH and FSH from
gonadotropes is sexually dimorphic, and in females,
the circulating hormone levels fluctuate dramatically
throughout the menstrual cycle. This differential
regulation is the result of a complex interplay of a
multitude of physiological inputs, including metabolic
status, stress, and inflammatory cues, in addition to
steroidal and peptide feedback mechanisms into the
hypothalamus and pituitary (–). GnRH is released
in a pulsatile manner (, ), and it has long been
known that each pulse of GnRH results in a con-
comitant pulse of LH transcription and secretion ().
Pulses of FSH can also be detected following GnRH
stimulation of gonadotropes, but in contrast to LH,
this regulation appears to be at the level of FSH
b-subunit transcription, as FSH secretion appears to
be largely constitutive (–). The issue of how GnRH
differentially regulates the contrasting profiles of the
two gonadotropins across the menstrual cycle was
resolved following the discovery that LH and FSH
transcription was differentially affected by GnRH pulse
frequency (, ). High-frequency pulses of GnRH
appear to favor LH, whereas low-frequency pulses
promote FSH synthesis.
Gonadotropin hormone structure and function
The pituitary gonadotropin hormones LH and FSH, as
well as the placentally derived LH paralog hCG, are all
large, heterodimeric glycoproteins that consist of a-
and b-subunits. Along with the pituitary-derived TSH,
this group of hormones is termed the glycoprotein
hormones. The glycoprotein hormone heterodimers
are composed of a -amino acid (aa) a-subunit
encoded by a single gene common to all three hor-
mones () and a hormone-specific b-subunit (LH:
 aa; hCG:  aa; FSH:  aa; and TSH:  aa).
LH and hCG differ only by a C-terminal, -aa peptide
[carboxy-terminal peptide (CTP)] extension in hCG,
doi: 10.1210/er.2018-00052
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and both activate the LHR with similar potencies in
vitro (). However, the CTP confers an increased half-
life to hCG. The a- and b-subunit heterodimers form
through noncovalent interactions, whereas the intra-
subunit tertiary structure is largely governed by
disulfide bonds (), with the a- and b-subunits
containing  and  cysteine residues, respectively
(). The resolution of the crystal structure of hCG
demonstrated that the a- and b-subunit interaction is
stabilized by a cysteine loop of the b-subunit that
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encircles loop two of the a-subunit in a manner
resembling a seatbelt (). Two disulfide bonds,
conserved in all of the human glycoprotein hormones,
are responsible for the “locking” of the seatbelt around
the a-subunit (). Whereas the a- and b-subunits
both contribute to receptor binding, molecular
modeling and the use of chimeric proteins have
identified a “determinant” loop in the b-subunit (aa 
to ) that confers receptor binding specificity
(–).
The mature gonadotropin hormones are heavily
glycosylated. The common a-subunit has two N-
linked glycosylation sites (N and N), with the
hormone-specific b-subunits containing either one
(LH: N) or two (FSH: N and N and hCG: N
and N) N-linked sites (). The hCG CTP extension
also contains an additional four O-linked glycosylation
sites. Chemical and enzymatic removal of the oligo-
saccharide moieties or removal of a- and b-subunit
glycosylation sites via site-directed mutagenesis has
revealed a number of important roles for these resi-
dues. Whereas the function of O-linked glycosylation
of the CTP appears to be predominantly related to
reduced clearance rates and increased half-life and
biological activity (, ), N-linked glycosylation
appears to be more complex. Evidence for roles in
determining clearance rates has been reported, with a
number of studies suggesting that the number of
terminal sialic acid or sulfonated N-acetylgalactos-
amine residues present on the glycan determines the
rate of hepatic clearance, with increased sialylation
reflecting a longer half-life and increased sulfonation
resulting in faster clearance (–). Roles in receptor
activation have also been demonstrated, as deletion of
the N-linked glycosylation residues reduces biological
activity with only a marginal effect on receptor binding
(, –). Mutagenesis studies have demonstrated
that removal of the a-subunit N site affects bio-
activity (, –), whereas loss of b-subunit gly-
cosylation sites may exacerbate the loss of bioactivity
induced by loss of N (). Roles of glycosylation in
subunit synthesis and heterodimer secretion have also
been indicated (, , , ).
The nature, as well as the presence, of the glycosylation
also appears to affect gonadotropin bioactivity. Charge
variants (due to differential glycosylation) and variants
with the same net charge but different glycosylation status
can affect biological activity. For example, basic isoforms
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(reduced sialylation) display higher binding affinities and
biopotencies in heterologous cell-assay systems than the
acidic isoforms, and some of the most basic isoforms
(pH . .) have antagonistic activity (–). In-
triguingly, antagonism of GnRHR has been shown to
result in elaboration of a greater proportion of these basic
forms of FSH, which act as antagonists (). Differentially
glycosylated FSH isoforms appear to have physiological
relevance, as during puberty and the transition from
early-to-late follicular phase, there is a progressive increase
in the ratio of basic-to-acidic isoforms, and this appears to
be tightly controlled by estradiol (). Moreover, different
FSH glycoforms are apparently capable of activating
different FSHR signaling modalities, with a number of
studies documenting differential recruitment of down-
stream signaling components (Gas, Gai, and b-arrestin)
and differences in cAMP release, estradiol production,
tissue-type plasminogen activator, cytochrome P
aromatase, and a-inhibin subunit expression (, , ,
, , ).
Gonadotropin receptor structure and function
The gonadotropin receptors are members of the
glycoprotein-hormone receptor subfamily of the
rhodopsin-like class of G protein–coupled receptors
(GPCRs) and have the prototypical seven trans-
membrane (TM) domain architecture of GPCRs. The
transmembrane (TM) domains are linked by three
intracellular loops and three extracellular loops (ECLs)
with an intracellular C-terminus and an extracellular
N-terminal domain (ECD). Ligand activation of
GPCRs promotes association with intracellular GDP-
bound heterotrimeric G proteins. The activated re-
ceptor acts as a guanine-nucleotide exchange factor,
promoting GTP displacement of GDP on the G
protein Ga-subunit. This exchange promotes disso-
ciation of the heterotrimeric G protein into a
monomeric GTP-Ga-subunit and a heterodimeric
Gbg-subunit. The dissociated subunits then activate
intracellular signaling pathways. In the case of the
gonadotropin receptors, the activated Ga-subunits are
responsible for activation of the enzyme adenylyl
cyclase, resulting in the production of the second
messenger cAMP. Whereas cAMP is acknowledged as
the “classical” intracellular signal, generated following
gonadotropin receptor activation, inositol phosphate/
Ca+, MAPK (ERK and p), and phosphatidylinositol
-kinase/AKT signaling modalities have also been
reported (–). Moreover, the phenomena of “biased
agonism,” whereby specific ligands preferentially recruit
certain signaling pathways, which is well described for
other GPCRs, has only recently emerged for gonado-
tropin receptors, predominantly through the use of
small molecule agonists (discussed later).
The glycoprotein hormone receptors are unique
among GPCRs in having very large ECDs (. aa),
consisting of a cysteine-rich domain, followed by
several leucine-rich repeats (LRRs) (, ). The
914 Anderson et al Peptide and Nonpeptide Gonadotropin Analogs
glycoprotein hormones interact with this large N-
terminal domain (). The LRRs have been demon-
strated to form a slightly curved solenoid, with the
hormone binding to the curved inner surface.
The receptor wraps around the central portion of the
hormone through both hormone subunits and occurs
over a large interface, described as a “hand clasp.” This
interaction is mediated via a number of charge in-
teractions, salt bridges, and aromatic ring-stacking
interactions (, ). A region proximal to the ECL/
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TM domains, which is designated the “hinge region,”
is known to play an important role in the activation of
the receptor following hormone binding. The mech-
anism through which activation occurs is not fully
known, but resolution of the crystal structure of FSH,
in complex with the entire ectodomain of FSHR
(including the hinge region), suggested a possible
mechanism via a second hormone-interaction site at a
sulfated tyrosine in the C-terminus of the hinge (Y)
(, ). This interaction is proposed to lift the hinge
region, leading to activation of the receptor through
the unmasking of an intramolecular agonist sequence
juxtaposed to the ECL/TM (Fig. ) (, ). This is in
contrast to previous hypothesized mechanisms,
whereby the hormone itself was proposed to make
direct contact with the ECLs and juxtamembrane
regions of the TM domains to induce receptor ac-
tivation ().
Gonadotropin Analogs in Clinical Use
The importance of the pituitary in reproductive
physiology was determined in the early th century.
The pioneering work of Bernhard Zondek in the
s and s linked the secretion of gonado-
tropins (then termed prolan A and B) from the
pituitary to follicular growth and steroidogenesis
(), paving the way for the application of gonad-
otropins in clinical medicine. To this end, several
gonadotropin analogs/preparations have been devel-
oped for clinical use. Indeed, urinary hCG (pregnyl)
has been available for therapeutic application from as
early as  ().
Purified/recombinant gonadotropin preparations
Initial preparations of gonadotropins for clinical use
were derived from pituitary extracts; however, this
practice was discontinued, as the supply of human
pituitaries could not meet the demand and also, the
recognition of a possible link to the transmission of
prion disease (). Endogenous gonadotropins are
now extracted from the urine of pregnant or post-
menopausal women, including chorionic gonadotro-
pin (hCG), urofollitropin (FSH), and menotropin/
human menopausal gonadotropin (hMG; which
contains both FSH and LH). These preparations
continue to be widely used, despite issues regarding a
Endocrine Reviews, December 2018, 39(6):911–937
 REVIEW

Figure 1. Two-step FSH/FSHR recognition and activation. The ECD LRRs of FSHR, in a putative orientation relative to the seven
transmembrane (7TM) domain, are shown as a magenta block with a hinge, and the 7TM domain is shown as a cylinder, with the
inactivated state colored gray and the activated state colored green. Sulfated Y335 is shown as a yellow ball, residue S273 is shown as
a green star, and disulfide bonds are shown as a brown stick. Heterotrimeric Gs or b-arrestin protein is shown as a green ellipsoid. FSH
heterodimer is represented in purple, with carbohydrates at N52a shown as a yellow, Y-shaped stick. (a) Inactive receptor is bound by
hormone, (b) resulting in Y335 in the receptor hinge lifting via interaction with the hormone, permitting (c) activation of the 7TM
domain. HBSD, hormone-binding subdomain; SSSD, signal-specificity subdomain. Reproduced with permission from Jiang X, Liu H,
Chen X, et al. Structure of follicle-stimulating hormone in complex with the entire ectodomain of its receptor. Proc Natl Acad Sci USA
2012; 109(31):12491–12496. [© 2018 Illustration Presentation ENDOCRINE SOCIETY]

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lack of regulatory control, batch-to-batch variation,
and limited source availability (Table ). However,
continued improvement of purification methodolo-
gies has enabled the production of highly purified
preparations.
The advent of the molecular era resulted in the
availability of recombinant gonadotropins [follitropin
alfa/beta/delta (FSH), lutropin alfa (LH), and cho-
riogonadotropin alfa (hCG) (Table )], allowing for
the production of highly pure preparations, batch-to-
batch consistency, and flexible dosing regimens and
protocols, resulting in more effective personalized-
treatment regimens. Responses to urinary and recombi-
nant gonadotropin preparations are similar (), and
because of the limited evidence of benefit in assisted
reproduction and associated cost implications, recombi-
nant gonadotropins have only partly replaced urinary
hCG/hMG in fertility treatments.
Different sources and preparations of gonadotro-
pins (both recombinant and human derived) can vary
in their receptor binding and signal-transduction
capabilities, and this is related to their glycosylation
status. For example, differences in the clinical activity
of different preparations have been attributed to
carbohydrate content, which varies depending on the
methods used for their production and/or purification
().
Gonadotropin chimeras (corifollitropin alfa)
In addition to enabling the production of large
quantities of native heterodimer, recombinant tech-
nologies have been used to generate chimeric go-
nadotropins, such as corifollitropin alfa (Org ;
Table  and Table ), an FSH heterodimeric protein in
which the CTP of hCGb is fused to the C-terminus of
the b-subunit of FSH to increase the biological half-
life. The chimeric b-subunit (FSH-CTP) does not
affect biosynthesis or secretion of the heterodimer in
cells used for production of recombinant protein.
Moreover, FSH binding to rat testes membrane
preparations and steroidogenic activity in granulosa
cells were retained (). Furthermore, the in vivo
potency (measured as increased ovarian weight and
granulosa cell aromatase activity following adminis-
tration of corifollitropin alfa to immature, estrogen-
primed rats) and biological half-life of corifollitropin
alfa were significantly increased compared with wild-
type FSH, whereas no LHR binding was detected ().
A phase I nonblind multicenter study followed, in
which hypogonadotropic hypogonadal (HH) men
were injected subcutaneously (SC), four times with
 mg corifollitropin alfa at -week intervals. No
serious adverse effects or generation of antibodies
against the protein were reported, and the elimina-
tion half-life for corifollitropin alfa of  hours was
considerably longer than that of follitropin beta
(Puregon;  to  hours) (). In a subsequent study,
in healthy females of reproductive age, a high dose of
oral contraceptive (Lyndiol) was administered to
suppress endogenous gonadotropin secretion, fol-
lowed by , , , or  mg SC injection of FSH-
CTP. As in the HH males, no antibodies against the
compound were detected, and increases in serum
inhibin B concentration and follicular growth (both
number and size of follicles) were observed in a dose-
dependent manner. The elimination half-life was
approximately twofold slower than follitropin alfa
(Gonal-F). Interestingly, the time to reach maximum

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Table 1. Commercially Available Gonadotropin Preparations

Hormone Source Generic Name Example Brand Names (Manufacturer)

hMG Urinary Menotropin EEMA-HMG (Corona Remedies)

Eventin/Eventin HP (Maneesh Pharmaceuticals)

GMH/GMH HP (Sun Pharma)

Gynogen HP (Sanzyme)

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Humegon (Organon)

Humog/Humog HP (Bharat Serums and Vaccines)

IVF-M/IVF-M HP (LG Lifesciences)

Materna HMG (Emcure)

Menopur (Ferring Pharmaceuticals)

Menogon (Ferring Pharmaceuticals)

Repronex (Ferring Pharmaceuticals)

Merional/Eigenorm/Meriofert (IBSA)

Normegon (Organon)

Ovulate-M (VHB Life Sciences)

Persinal (Serum Institute of India)

Puregraf (GUFIC)

ZyHMG/ZyHMG HP (Zydus Cadila)

Pergonal (Serono Laboratories)

FSH Urinary Urofollitropin Bravelle (Ferring Pharmaceuticals)

Endogen HP (Sanzyme)

EEMA-FSH (Corona Remedies)

Fertinex (Serono Laboratories)

Foliculin/Foliculin HP (Bharat Serums and Vaccines)

Foligem (VHB Life Sciences)

Follicare (GUFIC)

Follimon (LG Lifesciences)

Folliova (Sanzyme)

Fostimon/Fostipur/Altermon (IBSA)

Materna FSH (Emcure)

Metrodin (Serono Laboratories)

Metrodin HP/ Fertinorm HP (Serono Laboratories)

Neogentin (Maneesh Pharmaceuticals)

Orgafol (Organon)

Ovitrop (Sun Pharma)

Sitrodin (Serum Institute of India)

ZyFSH/ZyFSH HP (Zydus Cadila)

(Continued )

916 Anderson et al Peptide and Nonpeptide Gonadotropin Analogs Endocrine Reviews, December 2018, 39(6):911–937
 REVIEW

Table 1. Continued
Hormone Source Generic Name Example Brand Names (Manufacturer)

Follitropin alfa Gonal-F (EMD Serono/Merck Serono)

Bemfola (Finox Biotech)

Cinnal-F (CinnaGen)

EEMA-rFSH (Corona Remedies)

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Folligraf (Bharat Serums and Vaccines)

Ovaleap (Teva Pharma B.V.)

Recombinant Follitropin beta EEMA-rFSH (Corona Remedies)

Follistim/Puregon/Fertavid (Merck & Co./MSD)

Recagon (Organon India)

Follitropin delta Rekovelle (Ferring Pharmaceuticals)

FSH chimera Recombinant Corifollitropin alfa Elonva (MSD)

LH Recombinant Lutropin alfa Luveris (EMD Serono/Merck Serono)

FSH/LH mix (2:1 ratio) Recombinant Follitropin alfa:lutropin alfa Pergoveris (EMD Serono/Merck Serono)

hCG Urinary Chorionic gonadotropin Pregnyl (Merck & Co./MSD)

A.P.L. (Wyeth-Ayerst)

Brevactid (Ferring Pharmaceuticals)

Choragon (Ferring Pharmaceuticals)

Choriomon/Gonasi HP (IBSA)

Chorionic gonadotropin (Ferring Pharmaceuticals)

EEMA-HP (Corona Remedies)

Endocorion (ELEA)

Fertigyn (Sun Pharma)

Follutein (Bristol-Myers Squibb)

Hucog HP (Bharat Serums and Vaccines)

IVF-C (LG Lifesciences)

Materna-hCG (Emcure)

Novarel (Ferring Pharmaceuticals)

Ovipure HP (Bharat Serums and Vaccines)

Ovulate-C (VHB Life Sciences)

Ovutrig HP (VHB Life Sciences)

Profasi/Profasi HP (Serono Laboratories)

Proferti-C (CBC Pharma)

Provigil (Maneesh Pharmaceuticals)

Pubergen HP (Sanzyme)

Puretrig (GUFIC)

ZyhCG/ZyhCG HP (Zydus Cadila)

(Continued )

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Table 1. Continued
Hormone Source Generic Name
Recombinant Choriogonadotropin alfa
Bold text, originator brands; italic text, marketing withdrawn.
Abbreviation: MSD, Merck Sharpe & Dohme.
serum concentration was also slower than that de-
scribed for recombinant FSH, possibly as a result of
slower absorption (, –).
In , pregnancy and live birth after use of
corifollitropin alfa in an ovarian hyperstimulation
protocol were reported. In this phase II trial, a -year-
old woman, who had primary infertility and two failed
in vitro fertilization cycles, was injected with  mg
corifollitropin alfa SC. After  days, daily injections of
follitropin beta (Puregon;  IU), in combination
with a GnRH antagonist, were administered for  days,
after which final oocyte maturation and ovulation
were induced by administration of , IU hCG. Of
the  oocytes that were retrieved,  were fertilized via
intracytoplasmic sperm injection, and two embryos
were implanted, resulting in one live birth ().
Subsequent studies showed that pregnancy rates in a
cohort of  patients undergoing ovarian stimula-
tion with a GnRH antagonist protocol were almost
indistinguishable between women treated with one SC
injection of corifollitropin alfa ( mg) or  daily
injections of recombinant FSH ( IU). Additional
efficacy and dosing studies have demonstrated that
corifollitropin alfa injections have favorable pharma-
cokinetic profiles and comparable, or moderately
improved, pharmacodynamic responses compared
with recombinant FSH. This suggests that a single
weekly injection of corifollitropin alfa could replace
daily injections of recombinant FSH during ovarian
hyperstimulation protocols (–).
As a result of the extended duration of action of
corifollitropin alfa and the inability to control dose
after administration, there is a risk that ovarian hy-
perstimulation syndrome (OHSS; the development of
multiple luteinized ovarian cysts, leading to ovarian
enlargement and development of vascular hyper-
permeability) may be exacerbated in patients treated
with this long-acting gonadotropin analog. However,
adverse events, including OHSS, in two additional
double-blind randomized trials in healthy women
(Engage) and women of lower body weight (Ensure).
were of similar frequency to that observed in pa-
tients treated with recombinant FSH (, ). A
further phase III trial (Trust) that studied the effects of
repeated administration of  mg corifollitropin alfa
in up to three controlled ovarian hyperstimulation
cycles concluded that there was no immunogenic-
ity or drug-related hypersensitivity (). Although no
918 Anderson et al Peptide and Nonpeptide Gonadotropin Analogs
Example Brand Names (Manufacturer)
Sifasi (Serum Institute of India)
Ovidrel/Ovitrelle (EMD/Merck Serono)
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increased incidence of OHSS was reported in the
Engage, Ensure, and Trust trials (, , ), women
with polycystic ovarian syndrome (PCOS) or with a
history of OHSS had been excluded from these trial
cohorts. Therefore, the suitability of corifollitropin alfa
for higher-risk patients remains to be determined.
Following phase III trials, in early , corifolli-
tropin alfa (under the trade name of Elonva; Table )
was approved for use in the European Union in
women undergoing controlled ovarian hyperstimu-
lation with a GnRH antagonist-assisted reproduction
protocol who have no history of OHSS or PCOS.
Therapeutic Applications of
Gonadotropin Analogs
Gonadotropin therapy in men
The differential sites of action of LH and FSH at Leydig
and Sertoli cells, respectively, underpin their clinical
use in the support of steroidogenesis and of sper-
matogenesis in men suffering from HH. hCG
provides a very convenient form of LH-like activity
and is generally administered at doses of  to 
IU, twice per week, with the opportunity to titrate the
dose, depending on the serum testosterone level
achieved. These doses are, however, far higher than
those needed to normalize intratesticular testosterone
concentrations in men with induced experimental
gonadotropin deficiency. Thus, low doses of hCG (
IU) on alternate days can normalize serum testos-
terone levels (). FSH (either urinary or recombinant)
is conventionally administered in addition to hCG.
However, in adult men with previously normal
spermatogenesis who have subsequently become
gonadotropin deficient, for example, as a result of
pituitary surgery, hCG alone can often restore
spermatogenesis. On the other hand, men who have
never achieved spermatogenesis also require FSH.
Whereas overall, the prospects for spermatogenesis
are high (~%) in men with either prepubertal- or
postpubertal-onset HH, it is more successful in men
with postpubertal onset (% vs %), with higher
sperm concentrations also being achieved (. vs .
million/ml). Prior androgen therapy was believed to be
associated with a slower response to gonadotropin
therapy (), but a meta-analysis of  studies revealed
no association (). However, testicular volume is a
Endocrine Reviews, December 2018, 39(6):911–937

positive prognostic indicator of response, as is previous
treatment with gonadotropins, whereas cryptorchi-
dism is an important negative predictor. The long-
acting version of FSH, corifollitropin alfa, has also been
used in hypogonadotropic men, and in a small open
label trial, spermatogenesis was established in % of
men who remained azoospermic after treatment with
hCG alone (). Administration of this long-acting
analog, once every  weeks, greatly reduces the
treatment burden, and on the basis of this limited
evidence, it appears similarly efficacious to FSH.
It is important to recognize that quantitatively,
normal spermatogenesis is often not achieved in these
cases and additionally, that significant duration of
treatment may be required before a detectable sperm
concentration is achieved. Thus, in one large study, the
median time to achieve the first detectable sperm was
. months (% confidence interval, . to .), with
conception achieved after a median of . months
(. to .). The median sperm concentration at
natural conception was . million/ml, with the lowest
sperm concentration at conception at . million/ml
(). Thus, men should be advised that treatment may
be required for at least  years. Moreover, the age of the
female partner can be an important determinant of
successful pregnancy. A commonly used protocol is
initiation of androgen production with the use of hCG
alone for ~ months and addition of FSH at that stage
unless sperm are already detectable in the ejaculate.
The main advantages of this approach are to save on
the cost of FSH and also, to reduce the frequency of
injections, from the five required for administration of
both gonadotropins, to two per week for just hCG. It is
possible that administration of both gonadotropins
from the start may result in more rapid onset of
spermatogenesis, but robust data supporting this are
absent. Additionally, pretreatment with FSH alone has
been proposed, and in men with congenital HH
without prior gonadotropin treatment, a small trial
indicated that this approach could be successful ().
FSH treatment of  months before GnRH (thus, in-
ducing both LH and FSH secretion) resulted in
normalization of serum inhibin B, increased testicular
volume, proliferation of Sertoli cells, and spermato-
gonia (at biopsy) and the presence of sperm in the
ejaculate in all men.
In adolescent boys with delayed puberty as a result
of HH, the standard approach of using testosterone
administration results in growth, virilization, and
psychosexual maturation without testicular develop-
ment or induction of fertility. Therefore, there is a
possibility that subsequent fertility may be improved if
gonadotropins are instead used for this purpose. A
recent multicenter study of  patients addressed this.
The administration of hCG with recombinant FSH
in boys and adolescents with HH resulted in the
presence of sperm in the ejaculate in over % of
subjects, whether they had previously been treated
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with testosterone and with similar final testicular
volumes (). Thus, in keeping with a meta-analysis in
adult men (), previous testosterone therapy was not
found to be disadvantageous, although a possible
advantage of gonadotropin-induced puberty would be
that spermatogenesis can subsequently be more rap-
idly induced in men previously treated with gonad-
otropins when fertility is desired.
Gonadotropin therapy in women
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Gonadotropins can be used to induce ovulation, either
to stimulate mono-ovulation (for support of natural
conception or intrauterine insemination) or in ARTs,
where a multifollicular response is desired (conven-
tionally called “controlled superovulation,” albeit with
less control than that term indicates).
Mono-ovulation
The ability of exogenous gonadotropin preparations to
induce ovulation in both hypogonadotropic women
and in those with normogonadotropic anovulation
(generally as a result of PCOS) has been recognized, as
such preparations became available in the late s
(). This approach excludes the normal, negative-
feedback pathways that fine tune LH and FSH se-
cretion from the pituitary to ensure selection and
ovulation of just one follicle from many antral follicles
present in the ovary at the start of the menstrual cycle.
To minimize multifollicular development and the
resultant risk of a multiple pregnancy, the current
regimen of choice is the low-dose step-up protocol,
wherein low initial doses of gonadotropins (generally
 to  IU FSH) are increased only at infrequent
intervals, initially after  weeks and thereafter, at
-week intervals (). A step-down protocol, aiming to
mimic more closely physiologic changes in FSH, has
also been developed with good success rates () but
has not been widely adopted.
Recombinant/urinary FSH is generally used for
women with PCOS (as they are not LH deficient) who
are resistant to antiestrogen therapy or who have
become ovulatory with antiestrogen but have not
conceived. Recent meta-analyses and guidelines con-
firm the value of gonadotropins as the appropriate
second-line therapy in women with PCOS wishing
to become pregnant (–), and whereas it may
offer a more rapid time to pregnancy than with clo-
mifene (), the complexity (and expense) of treat-
ment generally precludes this. With this approach,
mono-ovulation can be induced in over % of cycles,
with a live birth rate of ~% per cycle and a cu-
mulative live birth rate of ~%. This can be achieved
with a very low multiple live-birth rate of .% but only
by application of rigorous cancellation criteria in the
event of a multifollicular response ().
In severely hypogonadotropic women, prepara-
tions containing LH activity must be used. In such
patients, induction of ovulation is more difficult
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(with a higher cycle cancellation rate reflecting a higher
multifollicular response), and a lower pregnancy rate is
achieved. Therefore, such women are ideally treated
with pulsatile GnRH (whereby the involvement of
physiological feedback pathways contribute to high
rates of mono-ovulation), but this is of very limited
availability.
Superovulation
The use of high doses of FSH for controlled super-
ovulation before assisted reproduction has now
resulted in several million live births around the world.
There are several protocols described and well
established, and dosage is often guided by measure-
ment of antral follicle count or of serum anti-
Müllerian hormone, which can identify women at risk
for excessive or low response, with dose adaptation or
additional counseling as appropriate (, ). This
continues to be an evolving field reflecting limited
evidence comparing key aspects of treatment, such as
the optimal dose of FSH to optimize the response (,
). It does, however, carry some risk, particularly of
OHSS and of multiple pregnancy if more than
one embryo is subsequently transferred (, ).
The risk of OHSS has been dramatically reduced in
recent years by using GnRH antagonist-based
protocols to suppress endogenous gonadotropin
secretion rather than GnRH agonists that cause an
initial “flare” in GnRHR activity (, ). Whereas
GnRH antagonist protocols may be associated with a
slightly reduced live-birth rate, possibly as a result
of as-yet underoptimized luteal replacement, this
seems not to be the case in women with PCOS, and
it has been suggested as standard therapy for
that group (). GnRH antagonist cycles do not,
however, completely prevent the risk of the devel-
opment of OHSS, and other established thera-
pies include cycle cancellation, the use of a GnRH
agonist to trigger oocyte maturation, and a freeze-all
strategy, with the latter often referred to as “cycle
segmentation” ().
The risks of multiple pregnancies have been re-
duced in recent years by using elective single-embryo
transfer (). Although this results in reduced live-
birth rates per cycle, cumulative rates over multiple
cycles are maintained ().
Novel approaches to assisted reproduction using
gonadotropins have, in large part, been stimulated by
the need for protocols that do not require any delay
before their commencement, generally known as
random start protocols, and the double-stimulation
approach, which allows two stimulation cycles to be
administered with only a very brief interval in between.
Both of these approaches are of particular benefit in
patients newly diagnosed with cancer, where time is of
the essence, and normal endometrial development is
irrelevant, as all oocytes or resulting embryos will be
cryopreserved.
920 Anderson et al Peptide and Nonpeptide Gonadotropin Analogs
The initial approach to more rapid-onset FSH
administration involved luteolysis with the use of a
GnRH antagonist (), but it subsequently became
clear that FSH administration could be started at al-
most any stage of the cycle, other than in the im-
mediate periovulatory period. This approach gives
comparable oocyte number and apparent de-
velopmental competence to those obtained after
routine ovarian stimulation, and whereas data on
pregnancy rate remain limited as a result of the nature
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of the circumstances of these patients, they appear
acceptable (). The addition of the antiestrogen
tamoxifen and more recently, the aromatase inhibitor
letrozole to such regimens in women with hormone-
dependent cancer to reduce estradiol production has
also been used as a further level of refinement under
those specific circumstances (–), although this
use is “off label.”
Double stimulation is an alternative approach,
wherein gonadotropin administration is restarted a
few days after a GnRH agonist trigger (). This
approach can substantially increase the number of
oocytes obtained, and indeed, it may be the case that
more oocytes are obtained during the second phase of
stimulation than in the first, thus more than doubling
the oocyte yield (). This approach can also po-
tentially reduce the cancellation rate, particularly in
women with a poor ovarian response or who are older,
and the limited data that are available indicate that the
developmental potential of embryos derived from each
of the two stimulations is comparable (, ).
Development of Gonadotropin Analogs
Polypeptide gonadotropin analogs
Single-chain and dual-activity gonadotropins
As discussed, gonadotropin preparations (both en-
dogenous and recombinant) are used therapeutically
in a number of clinical settings. The duration of
treatment (in some cases, months) has necessitated the
development of long-acting polypeptide analogs to
mitigate the need for multiple injections for improved
patient convenience. Corifollitropin alfa is one ex-
ample of a long-acting FSH analog, although a major
drawback is that the corifollitropin alfa is a hetero-
dimer. The stability of corifollitropin alfa and heter-
odimeric urinary gonadotropins is an issue during
purification, shipping, and storage. In an attempt
to overcome these issues, Sugahara et al. ()
synthesized a single-chain hCG analog in which the
hCGb-subunit was fused to the N-terminus of the
gonadotropin a-subunit (hCGba; Table ). The in
vivo biopotency of this single-chain analog was shown
to be greater than the heterodimer, as measured in a
rat ovulatory assay (). The enhanced biological
activity was suggested to be a result of improved
Endocrine Reviews, December 2018, 39(6):911–937

pharmacokinetics, as the single-chain analog is not
subject to subunit dissociation (). Interestingly, the
single-chain analog with the a-subunit oriented at the
N-terminus of the b-subunit was secreted but inac-
tive, with no receptor binding or activity measured in
vitro, whereas orientation of the b-subunit at the N-
terminus of the a-subunit resulted in improved se-
cretion rates and permitted intramolecular interactions
similar to the native heterodimer, in addition to com-
parable in vitro IC and EC values ().
These findings with single-chain hCG led to the
development of single-chain analogs of LH and FSH
[(LHba, LHbDa (in which the terminal  aa of LHb
were removed), LHbD-CTP-a, LHb-CTP-a, and
FSHb-CTP-a; Table ], which had comparable or
improved in vitro secretion and biological activity
comparable with the native heterodimers (, , ).
Again, these single-chain analogs were engineered
with the b-subunits oriented at the N-terminus of the
a-subunit, using the hCGb CTP sequence as a linker.
The CTP contains multiple proline and serine residues
and is predicted to lack substantial secondary struc-
ture, thereby facilitating folding and native inter-
subunit heterodimer-like interactions. Additionally,
the O-linked glycosylation sites of the CTP reduced
hepatic clearance rates, contributing to increased serum
half-life and increased biopotency of the single-chain
analogs. The pharmacokinetic and pharmacodynamic
profiles of FSHb-CTP-a have been determined in
Rhesus monkeys by SC injection or IV bolus ( IU/kg).
The absorption half-life of FSHb-CTP-a was three-
fold slower than recombinant FSH following SC in-
jection, and the elimination half-life was increased
twofold or fourfold over recombinant FSH when ad-
ministered SC or IV, respectively (). The same study
also demonstrated that a single SC dose of the single-
chain analog could stimulate estrogen production for
 to  days ().
In vivo biopotency of the single-chain analogs of
FSHb-CTP-a and hCGba were also tested in sheep
passively immunized against GnRH, to suppress en-
dogenous gonadotropin secretion. Concurrent ad-
ministration of the single-chain analogs ( IU/kg IV),
followed by  IU hCG IV after  days to induce
ovulation, resulted in elevated serum progesterone and
the presence of corpus lutea, highly suggestive of
successful ovulation. Neither analog promoted estra-
diol production in isolation but dual administration
resulted in a robust estradiol increase,  days post-
injection (), therefore confirming that the single-
chain analogs have in vivo biological activity. This
study, demonstrating that the analogs could be ad-
ministered in combination to induce estradiol pro-
duction and folliculogenesis, led to the development
of dual-active, single-chain gonadotropin constructs,
including FSHb-CTP-CGb-a, FSHb-CTP-LHb-
CTP-a, and CGb-FSHb-CTP-a (Table ), with both
FSHR and LHR activities (, , ). The constructs
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FSHb-CTP-CGb-a and FSHb-CTP-LHb-CTP-a
were administered to sheep using the same experi-
mental design to test the single-chain analogs. Both
dual-active constructs elicited increases in serum es-
tradiol concentration,  to  days after administration,
similar to that stimulated by dual injection of the two
single-chain gonadotropin constructs. In addition, an
increase in ovarian weight (attributed to increased
numbers of follicles) and the presence of corpus lutea
were observed (, ). The clearance rate of the dual-
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active construct FSHb-CTP-LHb-CTP-a was also
significantly improved compared with endogenous
ovine gonadotropin ( to  hours) (, ).
Despite these promising indications, to date, single-
chain or dual-active gonadotropin analogs remain
untested in humans.
Differentially glycosylated gonadotropin analogs
Another strategy used to produce gonadotropins with
altered signaling/pharmacokinetic properties was the
production of differentially glycosylated analogs. The
addition of an N-terminal extension sequence to in-
troduce two additional N-linked glycosylation sites
(ANITVNITV) to the glycoprotein a-subunit of FSH
was found to increase its half-life, three- to fourfold
compared with FSH in a rat model (). This analog
(N-a FSH; Table ) exhibited lower in vitro–specific “To date, single-chain or dual-
activity than FSH but had higher in vivo potency active gonadotropin analogues
(measured by ovarian weight and increased estrogen remain untested in humans.”
production in rats), likely as a result of its increased
half-life. A single dose was as effective as daily doses of
the same concentration of FSH ().
Single-chain analogs, which have substitution of
the CTP linker with a synthetic linker containing two
N-linked carbohydrate signal sequences (N linker),
have also been demonstrated to increase serum half-
life in rats, with comparable in vitro and in vivo po-
tencies to those of analogs containing the CTP. A
single-chain FSH construct with this substitution
(FSHb-N-a; Table ) had comparable activity in vitro
to recombinant FSH. Its half-life was also similar to
FSHb-CTP-a and approximately twofold greater than
recombinant FSH in immature female rats after a
single IV injection. Rats administered with FSHb-N-
a also had higher mean ovarian weights,  days after
administration, compared with those administered
with recombinant FSH ().
A linker with N-linked glycosylation sites is ad-
vantageous, as in contrast to O-linked sites (such as
those of the CTP), there is a defined N-linked gly-
cosylation consensus sequence that can be exploited to
generate differentially glycosylated hormone variants
(). Analogs with varying numbers of N-linked
glycosylation sites (FSHb-N-a and FSHb-N-a)
between the subunits (Table ) have been pro-
duced to determine effects on biological half-life and
biopotency. Whereas elimination half-life in female
rats was comparable between analogs containing the
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N linker (FSHb-N-a) or CTP (FSHb-CTP-a)
linkers, there was only a modest improvement in
clearance rates for the FSHb-N-a analog (). In-
terestingly, despite a plateau in elimination half-life
and comparable augmentation of ovarian weight
following injection of FSHb-CTP-a, FSHb-N-a, and
FSHb-N-a analogs, there were increases in the
number of antral follicles and substantial increases in
inhibin A following treatment with all the N-linked
analogs (). The in vivo bioactivities of FSHb-N-a
and FSHb-N-a have since been examined further,
with -week-old female mice given a single IP injection
of  IU of the N-linked analogs, recombinant FSH, or
pregnant mare’s serum gonadotropin (PMSG; which
has activity at both FSHR and LHR in nonequine
species). The bioactivities of FSHb-N-a and FSHb-
N-a, measured by ovarian and uterine weights, in-
hibin A production, and a number of antral follicles,
superseded the recombinant FSH responses. PMSG
performed best in all measured parameters as a result
of its activities at both LHR and FSHR. However, the
supplementation of the FSHb-N-a and FSHb-N-a
injections with daily injections of . IU hCG resulted
in responses comparable with that of PMSG (). In
another mouse study,  IU IP of FSHb-N-a, FSHb-
CTP-a, or PMSG was administered, followed by  IU
hCG IP,  hours later. Increased ovulation and
embryo maturation were observed with FSHb-N-a
when compared with FSHb-CTP-a (. 6 .
embryos vs  6  embryos, respectively) (). The
delivery rates and litter sizes were comparable between
PMSG and FSHb-N-a and were higher or compa-
rable with recombinant FSH treatment ().
Again, although promising in vivo results have
been obtained for these differentially glycosylated
analogs, they have yet to be developed further for
clinical use.
Gonadotropin conjugates and suspensions with
increased half-life and bioavailability
Alternative methods to try to improve gonadotropin/
glycoprotein hormone pharmacokinetic properties
have included attachment of synthetic polymers to
increase half-life and bioavailability and therefore,
biological activity. Covalent addition of polyethylene
glycol (PEG; PEGylation) results in many advanta-
geous properties, such as increased solubility of hy-
drophobic molecules, increased size (reduced renal
clearance), reduced immunogenicity, and protection
from proteolytic degradation. Injection of Sprague-
Dawley rats with . mg/kg IM of a recombinant
PEGylated TSH analog resulted in a -fold increase in
plasma half-life and elevated thyroxine (T) levels for
up to  hours, with only moderate effects on receptor
binding affinity (). FSH has also been PEGylated
via covalent amide linkage (Table ), and this analog
retained FSH activity, as measured by progesterone
secretion of cultured bovine cumulus cells (), and
922 Anderson et al Peptide and Nonpeptide Gonadotropin Analogs
also had greater bioavailability compared with the
non-PEGylated control in rats ().
Another strategy for increasing FSH half-life was
the creation of a fusion-protein consisting of FSH a-
and b-subunits fused to a binding partner (e.g., im-
munoglobulin Fc fragments) of the neonatal Fc
receptor (FcRn). FSH fusion proteins containing
immunoglobulin Fc fragments had increased stability
and serum half-life. In addition, these fusion proteins
are protected from endosomal degradation by binding
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to the FcRn, facilitating recycling (–). When
female rats were injected SC with  nmol/kg
recombinant FSH or a heterodimeric FSH molecule
conjugated to an immunoglobulin Fc fragment (FSH-
Fc; Table ), ovarian weight was increased twofold in
the FSH-Fc-treated rats when compared with the
recombinant FSH-treated group ().
Modification of hormones by conjugation to en-
dogenous molecules, such as FcRn binding partners,
may result in advantageous properties, in addition to
increased half-life, such as oral/pulmonary bio-
availability. For example, fusion proteins comprising
FcRn binding partners can be shuttled across epithelial
barriers, opening the intriguing possibility of creating a
gonadotropin analog that can be administered via
nasal or pulmonary routes using aerosols. This has
been demonstrated in the cynomolgus monkey, with a
single pulmonary dose of heterodimeric FSH-Fc (
mg/kg), eliciting a -fold increase in serum inhibin B
levels (). Conjugation of peptidic hormones and
large macromolecules to vitamin B has also been
shown to confer oral bioavailability. Vitamin B
interacts with an intrinsic factor, which in turn, binds
to its cognate receptor located on the terminal ileum of
the small intestine and is internalized by endocytosis.
Vitamin B conjugation to macromolecules, such as
gonadotropins with poor oral bioavailability, may
therefore improve uptake via oral administration.
Whereas this method has not yet been applied to
gonadotropins, a D-lys GnRH analog has been suc-
cessfully crosslinked to monocarboxyl-vitamin B,
and this conjugate has been shown to retain bioactivity
when administered orally (PO) in a mouse ovulation
assay ().
Another approach to increase hormone half-life
was the development of sustained-release formula-
tions. Single injections of porcine FSH dissolved in an
aluminum hydroxide gel suspension were used to
stimulate folliculogenesis in both rabbits and cows
(, ). The number of oocytes recovered and the
number fertilized following a single injection of the
aluminum hydroxide adjuvant were not statistically
different than those treated with daily injections of
porcine FSH dissolved in saline, and measurement of
serum FSH concentrations indicated that the levels of
hormone were sustained throughout the treatment
period, confirming its sustained release (, ). In
rabbits, the aluminum hydroxide gel preparation was
Endocrine Reviews, December 2018, 39(6):911–937

compared with one of FSH dissolved in an alternative
adjuvant, polyvinylpyrrolidone, but superovulation
was found to be much more effective when FSH was
administered in the aluminum hydroxide preparation
().
Whereas these modified analogs with improved
bioactivity/bioavailability have not been tested clini-
cally, they do offer an exciting possibility for possible
future development of improved or less invasive go-
nadotropin therapeutics.
Polypeptide-gonadotropin antagonists
In addition to the development of polypeptide-based
gonadotropin preparations/analogs for activation of
the gonadotropin receptors, a number of strategies
have also been used in an attempt to generate
polypeptide-based gonadotropin antagonists for po-
tential application in contraception. Fusion of two
hCG b-subunits as a single chain (hCGbb; Table )
resulted in an analog able to bind to the LHR (albeit
with a binding affinity lower than hCG;  and  nM,
respectively), which exhibited a dose-dependent in-
hibition of hCG actions (measured by decreased se-
rum testosterone concentration) in a rat model ().
As a result of the importance of glycosylation for
gonadotropin function, it has also been proposed that
deglycosylated gonadotropins, which retain receptor
binding activity but with reduced bioactivity (see the
“Gonadotropin hormone structure and function”
section), could also act as competitive antagonists with
therapeutic potential (, , , ). Deglycosyla-
tion can disrupt dimerization of the gonadotropin
subunits, but this can be overcome by the addition of
specific dimerization sequences () or by use of
single-chain hormones in which the two subunits are
covalently joined (see the “Single-chain and dual-
activity gonadotropins” section). Indeed, treatment
with  IU of a single-chain version of partially
deglycosylated hCG (Table ) has been shown to
reduce steroid hormone production and ovulation in
rats pretreated with  IU PMSG when compared with
stimulation with  IU hCG (). In addition, factors
associated with OHSS, such as vascular permeability
and expression of vascular endothelial growth factor,
were reduced in these animals, suggesting that this
analog could ameliorate the effects of prolonged ex-
posure to hCG after ovulation has been triggered in
patients with a high risk of OHSS (). However, there
are conflicting opinions regarding the antagonistic
properties of the deglycosylated hormone, with one
study showing no decrease in progesterone levels when
the deglycosylated hormone was administered to
healthy females in the midluteal phase, although the
authors acknowledged that this may be a result of the
presence of “spare receptors” and the inability to
achieve sufficient serum concentrations of the an-
tagonist (). In addition, chemical methods of
deglycosylation, such as hydrogen fluoride treatment,
doi: 10.1210/er.2018-00052
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often result in incomplete deglycosylation, which may
confound the interpretation of data ().
Nonpeptidic Low-Molecular-Weight
Gonadotropin Receptor Agonists
and Antagonists
Whereas the development of polypeptide gonado-
tropin analogs with increased serum half-life/bioactivity
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(described earlier) shows promise of single injections to
replace the multiple injections of the natural hormones
used in ART, small-molecule/low-molecular-weight
(LMW) orally active agonists would avoid trauma of
injection and overcome problems of preparation uni-
formity (arising from posttranslational modification
of peptide-based analogs) and have greater com-
pound stability (). However, despite several drug-
development programs focused on identification of
LMW gonadotropin analogs, only three have entered
human trials, and none are commercially available.
Desirable properties of such LMW analogs would
include good oral bioavailability, high potency, high
specificity, and high lipophilicity (which would enable
interaction with allosteric sites within the TM domains
of the target receptors; see later). “Small molecule/LMW orally
In addition to application in ART, orally bioavailable, active agonists would avoid
small-molecule antagonists at gonadotropin receptors trauma of injection and
may have clinical application as novel oral contraceptives overcome problems of
with potentially greater specificity and therefore, reduced preparation uniformity.”
risk of side-effects, such as headaches, weight gain,
nausea, and increased risk of serious complications, such
as venous thromboembolism, which occurs with current
steroidal contraceptive drugs ().
LMW LHR agonists may also be used potentially
for women at risk for OHSS. This syndrome has been
linked to fertility treatments using hCG injections to
achieve oocyte maturation and ovulation. hCG has a
long serum half-life; therefore, LMW LHR agonists
with shorter half-lives may well circumvent/attenuate
the risk of OHSS. In addition, orally active LMW
compounds are more amenable to titrating dosage.
Despite the theoretical, therapeutic potential of
LMW modulators of the gonadotropin receptors, a
number of persistent problems have hindered their
development into clinically applicable drugs. Firstly,
the high degree of sequence conservation across the
glycoprotein hormone receptor family and other re-
lated receptors, such as the LRR containing GPCRs,
means that drug crossreactivity can be an obstacle.
Indeed, this has been an issue with some LMW an-
alogs. However, as the natural glycoproteins are highly
specific, there is no reason to believe that this cannot
be achieved with the LMW molecules. Secondly, the
glycoprotein hormones are large heterodimeric pro-
teins that interact with their cognate receptors via
multiple structurally conserved contact sites and ad-
ditionally, have a two-step binding and activation
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mechanism (Fig. ). Therefore, it is difficult to imagine
orthosterically binding LMW molecules that could
simultaneously occupy and induce the structural
changes within the glycoprotein hormone receptor
ECDs to attain full efficacy of activation with low
potency. However, this concern has been overcome
with allosterically binding LMW molecules (see later).
Thirdly, promising lead compounds, identified through
in vitro screening, have been found to have little/no
in vivo efficacy (), as is a common feature of small-
molecule development in the pharmaceutical industry.
GPCRs are amphipathic molecules, complicating
crystallization/structural analysis, but exciting ad-
vances in X-ray crystallography methodologies, in-
cluding the use of thermodynamically stabilizing
mutations and nanobody-based stabilization of GPCR
active states, have revealed new opportunities for novel
drug design (, ). One such avenue is that of
allosteric modulators of GPCR activity that interact
with the receptor at a site distinct from the orthosteric
ligand binding site (site of binding of the endogenous
ligand). Allosteric GPCR modulators generally fall into
three classes: allosteric agonists and positive and
negative allosteric modulators (PAMs and NAMs,
respectively). Allosteric agonists have agonist activity
in the absence of the orthosteric ligand, whereas PAMs
and NAMs are defined as molecules that either
potentiate or attenuate agonist-mediated responses,
respectively. This can be through modulation of
orthosteric agonist binding affinity or the ability of the
agonist-occupied receptor to interact with intracellu-
lar signal transducers. The targeting of allosteric sites
can be advantageous, as they are generally less evolu-
tionarily conserved and often contain unique structural
features, providing a platform for the development of
drugs with a greater degree of target specificity ().
Additionally, many allosteric modulators only have
activity in the presence of the cobound endogenous
ligand, with the advantage that GPCR activation is
limited to the spatiotemporal release of the endogenous
ligand (). This is especially relevant in the context of
many neuroendocrine signaling axes, which have cy-
clical patterns of hormone release.
Allosteric binding sites for LMW compounds have
now been described for a number of GPCRs. Through
the modeling of the gonadotropin hormone recep-
tor TM domains, using adenosine A receptor and
rhodopsin receptor molecular models, alongside a
number of biochemical assays, and studies using
chimeric receptors, it has been proposed that the
gonadotropin receptors have two allosteric sites: one
major and one minor, labeled P and P, respectively
() (Fig. ). These sites are located toward the ECL
half of the TM helices, close to the transition of TM-
to-ECL domains, with P located among TMs III, IV,
V, and VI and P among TMs I, II, III, and VII ().
A substantial number of structural scaffolds have
been described that can allosterically bind to the
924 Anderson et al Peptide and Nonpeptide Gonadotropin Analogs
gonadotropin receptors, and a number of cell-based
screening assays have been used over the last  years,
resulting in the identification of various lead
compounds/chemical scaffolds, with specific in vitro
cellular and in vivo physiological activity profiles.
The most frequently targeted of the glycoprotein
hormone receptors for LMW drug discovery is the
FSHR. This is perhaps unsurprising, given the multiple
injections of hormone necessary for ovarian hyper-
stimulation in assisted reproductive therapy protocols.
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However, although a number of promising LMW
compounds have been developed for both gonado-
tropin receptors, none are yet in clinical use. The
following sections will describe the development of
some of the most promising LMW compound classes
that have been identified to date. For more detailed
insight regarding the chemical properties of many of
these different compound classes, the reader is referred
to van Straten and Timmers ().
FSHR agonists
Piperidine carboxyamides
In , Serono (now Merck Serono) used a luciferase
reporter gene high-throughput screen to identify pi-
peridine carboxyamide derivatives with activity in
Chinese hamster ovary (CHO) cells stably expressing
FSHR. A lead compound with an EC of . nM was
identified and was subsequently shown to have poor
stimulation of estradiol in rat granulosa cells (EC .
mM). However, this initial finding was valuable in the
discovery of a number of chemical scaffolds with
improved FSHR agonistic activity ().
Thiazolidinones
Thiazolidinone (TZD) scaffolds have been used pre-
viously in a number of drug-discovery programs and
have excellent drug-like properties (–). Indeed,
low-potency FSHR agonists have been described that
contain thiazole core structures (). In , Affy-
max screened ., compounds from a large TZD
library for agonist activity at the FSHR using a lu-
ciferase reporter gene assay in CHO cells expressing
FSHR. The design of this library was based on data that
had identified a TZD compound with moderate ag-
onist activity at the FSHR ( to  mM). Several hits
with increased potency were identified from this
screen, including “compound ,” which was a partial
agonist (% relative to recombinant FSH stimu-
lation), with an EC of  nM (). Compound
development addressed synthesis issues regarding
stereoselectivity on the heterocyclic ring (the less active
trans isomer predominates over the desired cis isomer
during synthesis) and resulted in stable TZD com-
pounds containing g-lactam congeners and -alkyl
substituents with comparable potencies and efficacies
to the original lead compound (, ). Another
screen by Affymax and Wyeth (since acquired by
Endocrine Reviews, December 2018, 39(6):911–937

Pfizer) of a collection of combinatorial libraries con-
taining over , compounds and represent-
ing . core scaffolds identified two additional FSHR
agonists with TZD scaffolds (with potencies of ~ mM)
(). Parallel synthesis resulted in three compounds
with nanomolar potency. Subsequent studies using
chimeric receptors in which the N-terminal domain
and TM domains of the TSH receptor (TSHR) and
FSHR were interchanged indicated that these analogs
act via an interaction with the TM domains of the
FSHR (unlike the natural hormone that interacts with
the ECD). Further analysis using chimeric receptors
narrowed down the site of interaction to the area of the
receptor between TM I and ECL (). One of the
identified compounds, “compound ” (Table ), was
demonstrated to be a full agonist of the FSHR and
could induce progesterone production in mouse ad-
renal Y cells transfected with FSHR and estradiol
production in primary rat granulosa cells (), had
in vivo activity in a mouse cumulus expansion assay,
and demonstrated a dose-dependent increase in the
number of ovulated oocytes in a rat ovulation assay.
Although this compound had promising FSHR ac-
tivity, unfortunately, it had poor oral bioavailability,
and micronucleus screening assays identified geno-
toxic effects (–).
Wyeth/Affymax further characterized the pharma-
cological properties of several TZD compounds and
demonstrated that small modifications to the TZD ring
can result in completely different pharmacologies,
resulting in anything from agonists through to negative
modulators able to inhibit estradiol production in rat
granulosa cells via activation of Gai () (Table ).
Thus, a single TZD core structure has the potential to be
used to develop a spectrum of LMW allosteric mod-
ulators targeting multiple FSHR signaling pathways.
Diketopiperazines
A high-throughput screen in  by Pharmacopeia
(since acquired by Ligand Pharmaceuticals) and Or-
ganon [since acquired by Schering-Plough and then
merged with Merck & Co./Merck Sharpe & Dohme
(MSD)] of approximately two million compounds,
identified a number of biaryl agonists of the
FSHR with different substituents on the phenyl rings.
The most potent compounds contained heterocyclic
diketopiperazine substituents (). Lead optimiza-
tion, aimed at modification of the diketopiperazine
core side-chains, increased their potency from the low
micromolar to the low nanomolar range in both lu-
ciferase reporter gene and cAMP assays (). How-
ever, these compounds do not appear to have been
developed further, and there is no information re-
garding specificity or in vivo activity of these hit
compounds. At a similar time, Serono also filed a
patent describing substituted piperazine compounds
with FSHR agonistic activity in the low micromolar to
low nanomolar range ().
doi: 10.1210/er.2018-00052
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Hexahydroquinolines
Two patents filed in  by Organon detail the use
of hexahydroquinoline scaffolds (-phenyl--oxo-
l,,,,,-hexahydroquinolines) as FSHR activators
in vitro (, ). These patents describe several
LMW compounds with potencies in the low nano-
molar (, nM) range in an in vitro luciferase re-
porter assay. In , the same company published a
description of a hexahydroquinoline LMW PAM of
the FSHR, Org - (Table ). This molecule had
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agonist activity in a luciferase reporter assay and was
demonstrated to bind via a site distinct from the en-
dogenous hormone. In addition to the allosteric agonist
activity, coincubation with FSH demonstrated Org
- PAM activity, inducing a substantial (sixfold)
increase in FSH affinity, which translated to a fourfold
increase in FSH potency in the luciferase reporter assay
(). Org - was also demonstrated to induce
follicular growth in a rat ovulation assay when admin-
istered PO and thus, represented an orally active FSHR
LMW agonist with in vivo efficacy (). MSD has also
investigated similar compound scaffolds, including MK-
 (a dihydropyrroloisoquinoline) as LMW allosteric
FSHR agonists. However, when MK- was tested
in a dose-escalation study in healthy human females,
it was unable to confirm effects on follicular develop-
ment and found that it unexpectedly affected thyroid
function (), suggestive of activity at the TSHR, which
Figure 2. Top view of the LHCGR, with a partially clipped
backbone of the ECLs, visualizing the potential binding region
for small allosteric LMW ligands (magenta). This ligand-binding
region can be subdivided further into a major and a minor
pocket. TMH, TM helix. Reproduced with permission from
Heitman LH, Kleinau G, Brussee J, et al. Determination of different
putative allosteric binding pockets at the lutropin receptor by
using diverse drug-like low molecular weight ligands. Mol Cell
Endocrinol 2012;351(2):326–336. [© 2018 Illustration Presentation
ENDOCRINE SOCIETY]
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Table 2. Chimeric and Single-Chain Gonadotropin Analogs
emphasizes the importance of good selectivity margins
for these compounds.
Other agonists
MSD has also described two LMW FSHR agonists with
undisclosed scaffolds and interesting pharmacological
profiles. These agonists have nanomolar activity at the
FSHR ( to  nM) and display similar responses to
recombinant FSH in a luciferase reporter assay ().
These LMW FSHR agonists were unique in that they had
particularly short-acting profiles (half-life . to . hours,
time to reach maximum serum concentration . to .
hour). Intriguingly these compounds resulted in %
suppression of ovulation in cycling Orga rats at doses of
 mg/kg PO when administered twice daily. The
926 Anderson et al Peptide and Nonpeptide Gonadotropin Analogs
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inhibition of ovulation resulted from the formation of
luteinized, unruptured follicles, and this effect was re-
versible, with spontaneous ovulation recovery following
discontinuation of treatment (). These results could
not be fully replicated in mono-ovulatory species (cyn-
omolgus monkey) (), but this still represents an
approach to the development of a reversible female
contraceptive.
FSHR antagonists
Sulfonic acid-containing compounds
The sulfonic acid-containing compound, suramin, has
been used as an IV antiparasitic for over  years
(). In addition to its antiparasitic properties,
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927
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Table 3. Selected LMW Gonadotropin Analog Structures

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suramin has been demonstrated to inhibit the sig-
naling activity of a number of peptide hormones,
including platelet-derived growth factor, epidermal
growth factor (), and FSH (, ). Subsequently,
suramin has been shown to compete with FSH for
binding to the FSHR-ECD (). Interestingly, a
second mechanism of inhibition may involve dis-
ruption of certain GPCR-G protein interactions (,
). In a cohort of male patients receiving suramin
as a therapy for refractory cancer, all showed a marked
decrease in plasma testosterone, and this effect
was mirrored in a rat Leydig cell assay (). The
dramatic in vivo effect on testosterone secretion,
combined with lack of specificity (including docu-
mented LHR interaction) (, ), precipitated
the development of other LMW FSHR antagonists
containing sulfonic acid moieties (, ). Com-
pound screening by Wyeth identified three (bis)
sulfonic acid, (bis)benzamide FSHR antagonists with
moderate potencies of cAMP inhibition, estradiol
inhibition (granulosa cell assay), and progesterone
inhibition (in an adrenal Y cell assay) and IC
values in the low micromolar range (). Unlike
suramin, these compounds displayed marked im-
provement in specificity, with no LHR binding ac-
tivity measured up to  mM and no inhibition of
TSHR cAMP response up to  mM (). Also in
, a napthalene sulfonic acid compound (“com-
pound ”; Table ) was identified by Wyeth through
library screening for compounds that inhibited FSH
binding to FSHR. The lead compound inhibited FSH
binding with an IC of . mM, and interestingly,
although it bound the FSHR ECD, the inhibition of
FSH binding was noncompetitive and led to a re-
duction in FSH binding sites but not affinity ().
This compound was also able to inhibit the FSH
cAMP response in a dose-dependent manner with
% efficacy, inhibited estradiol production in
granulosa cells, and when administered at  mg/kg
IP to cycling female rats, inhibited ovulation in %
of treated animals (). Analogs were developed
to examine structure-activity relationships, and a
number of substitutions were identified as critical for
activity, including the sulfonic acid moiety ().
Issues regarding off-target effects, safety, and oral
bioavailability have hampered the development of
these compounds for clinical application. In the in vivo
rat ovulation model, napthalene sulfonic acid induced
mild inflammation of the ovarian surface epithelium,
and the rats displayed a substantial retardation in
growth rate (). It is unclear whether these effects
may have indirectly contributed to ovulation in-
hibition. Furthermore, the oral absorption of the
(bis)sulfonic acid, (bis)benzamide compounds was
predicted to be poor, and the conversion of the sul-
fonic acid moieties to carboxylic acid, although pre-
dicted to improve absorption, abolished FSHR binding
activity ().
928 Anderson et al Peptide and Nonpeptide Gonadotropin Analogs
Aminoalkylamides
A substituted aminoalkylamide series of FSHR an-
tagonists has also been reported with a spectrum of
potencies. Two of the most potent compounds,
identified in a cAMP screening assay by Ortho-McNeil
Pharmaceutical, were tested for ovulation and
spermatogenic inhibitory effects in Wistar rats, but
following oral dosing at  mg/kg, only very limited
effects were noted (). In the absence of any
pharmacokinetic data, it is unclear whether the limited
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effects are a result of restricted oral bioavailability/poor
intestinal absorption/rapid clearance, as the two
compounds had activity in in vitro cAMP and gran-
ulosa cell assays.
Tetrahydroquinolines
Organon identified a tetrahydroquinoline (THQ)
scaffold with FSHR agonistic activity in a luciferase
assay screen, with a moderate potency of . mM and
an efficacy of % relative to FSH. This compound was
selected for lead optimization, and surprisingly, it was
discovered that introduction of an aromatic group at
position  of the THQ scaffold resulted in a switch
from full agonist to full antagonist, with nanomolar
IC values (compound ; Table ) (). Biphenyl
moieties were also tolerated, implying that the binding
pocket was large and lipophilic, with competition
binding assays determining that the pocket is likely to
be located in the TM domain (). In a mouse ex
vivo follicular growth assay, one THQ compound
with a biphenyl substituent was demonstrated to in-
hibit follicular growth in the presence of FSH and
inhibited ovulation in % of follicles ().
Benzamide derivatives
An homogenous time-resolved fluorescence screening
assay was used by Addex Pharmaceuticals to identify
FSHR NAMs and identified a benzamide compound
(ADX; Table ) with NAM activity (IC . mM
with % inhibition of FSH EC). This NAM was
ascertained to be a biased antagonist of the FSHR, as in
the presence of FSH, it inhibited cAMP and pro-
gesterone production in vitro in a dose-dependent
manner but failed to inhibit estradiol production
and at doses . mM, stimulated estradiol production
(). This finding was surprising and challenges the
paradigm that estradiol production is entirely de-
pendent on cAMP signaling. In an in vivo setting, SC
administration of ADX at  mg/kg resulted in
no differences in number of oocytes recovered and
ovarian weight of rats treated with  mg pituitary FSH
and ovulated with  IU hCG (). The authors of
this earlier article postulate that this may be a result of
the inability of ADX to suppress estradiol
production. However, women with PCOS have suc-
cessful pregnancies despite suppression of estradiol
with letrozole or tamoxifen, so suppression of estra-
diol may be necessary but not sufficient. Two additional
Endocrine Reviews, December 2018, 39(6):911–937

benzamide analogs were subsequently identified by
Addex, corroborating the description of biased antag-
onism identified for ADX and further sub-
stantiating the requirement for blockade of both arms of
the FSHR steroidogenic signaling pathway to inhibit
follicular maturation and ovulation in rats ().
Other FSHR antagonists
Pyrrolobenzodiazepine and acyltryptophanol LMW
FSHR antagonists were identified through luciferase
and homogenous time-resolved fluorescence screen-
ing assays, respectively, with IC concentrations in the
micro- and nanomolar ranges (, ).
LHR agonists
A number of LHR agonists have been developed by
the pharmaceutical industry.
Thienopyrimidines
Organon reported an orally active, nonpeptidic LMW
agonist for the LHR. In a high-throughput screen of a
compound library, using a luciferase reporter gene
assay, a thieno[,-d]pyrimidine was identified as
having agonist activity at the LHR (). A series of
thienopyrimidine and thienopyridine analogs were
subsequently synthesized and their in vitro activity
determined. The most potent LHR agonist identified
was the thienopyrimidine, Org  (EC of  nM)
(), which was found to stimulate testosterone
production in cultured mouse Leydig cells (EC of
. mM) and was able to stimulate ovulation in %
of urinary FSH-primed female mice, using a dose of
 mg/kg PO (). Org  has since been shown
to have partial agonist activity at the TSHR but with
~-fold lower potency and no substantial activity at
the FSHR (, ). With the use of computer-based
docking, a binding pocket was delineated among TMs
III, IV, V, VI, and VII and ECL, with the cleft formed
in the LHR larger than that of TSHR (, ). This
pocket was verified through mutagenesis of receptor
residues predicted to be of importance for Org 
interaction and by synthesis of analogs hypothesized to
increase specificity or ablate binding. Of particular
importance is a glutamate residue in TMIII that is
conserved between LHR and TSHR and is predicted to
form a hydrogen bond with the amino group of Org
 (, ). Mutation of this residue completely
ablated Org -mediated TSHR signaling. Fur-
thermore, an analog of Org  with addition of
“steric bulk” to the amide (an extra methyl group) was
sufficient to block Org -mediated TSHR sig-
naling but not LHR signaling, resulting in a substantial
improvement in specificity. This steric bulk was hy-
pothesized to inhibit insertion into the smaller TSHR
binding cleft but still allow accommodation into the
slightly larger LHR pocket (, ).
Further lead optimization of Org  led to the
development of another thienopyrimidine, Org 
doi: 10.1210/er.2018-00052
REVIEW
(Table ), which displayed nanomolar potency at the
LHR (EC of . nM; % efficacy compared with
LH), no activity at the TSHR, and reduced activity at
the FSHR (-fold reduced potency than at the LHR)
(). Interestingly, this study also highlighted biased
signaling properties of Org . Whereas LH ac-
tivation of the LHR stimulated both cAMP (EC 
pM) and phospholipase C (PLC; EC . nM), no
substantial activation of the PLC pathway by Org
 was measured, suggestive of biased agonism
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(). In addition, whereas Org  did not antag-
onize LH-induced intracellular cAMP accumulation,
it did strongly antagonize (% maximal inhibition) the
LH-induced activation of PLC in a noncompetitive
manner, suggestive of an allosteric binding mechanism
(). In support of this, Org  was shown to
reduce noncompetitively [I]hCG binding ().
Subsequent studies have both confirmed and re-
pudiated the described increase in [I]hCG dis-
sociation (, ), but nonetheless, the observed
inability of the LMW compounds to compete with
hormone binding supports the theory that these
compounds are acting in an allosteric manner.
The biological activity of Org  has since been
investigated more thoroughly. In an ex vivo ovulation-
induction assay using cultured mouse follicles,  mM “Issues regarding off-target
Org  was able to stimulate maximal levels of effects, safety, and oral
ovulation and progesterone production, similar to that bioavailability have hampered
seen using hCG, and was able to induce testosterone the development of these
production in primary Leydig cell cultures with high compounds.”
potency, although with lower efficacy than hCG ().
In vivo, Org  has been shown to have good oral
availability (% in rat and % in dog) with a short
half-life (. hours in rat and . hours in dog) ().
The occurrence of OHSS has been associated with the
long half-life of hCG (half-life ~ to  hours
depending on source and purification methodology)
(); therefore, the short half-life of Org  may
have appreciable advantages. Indeed, when Org 
was administered to PMSG-primed female rats, even
at high concentrations ( mg/kg), there was no
evidence of an increase in vascular permeability or
vascular endothelial growth factor levels, unlike that
seen with hCG or LH ().
In GnRH antagonist-treated rats, a single -mg/kg
dose of Org  PO resulted in ovulation of a
normal number of ova, comparable with the hCG
control ( IU/kg SC). Ova fertilization rate, im-
plantation rate, and subsequent fetal development
were all within normal parameters. In male rats at
concentrations $ mg/kg PO, similar levels of tes-
tosterone stimulation were measured to those pro-
duced after treatment with  IU hCG SC ().
Perhaps most promisingly, Org  and another
related thienopyrimidine, Org , have shown
efficacy in humans. Both were shown to induce
ovulation when administered PO in a preclinical trial
with healthy females. Org  was safe and well
https://academic.oup.com/edrv 929
REVIEW
tolerated up to a maximum dose of  mg ().
The GnRH analog, Ganirelix, was administered SC for
pituitary suppression in conjunction with recombi-
nant FSH to stimulate follicular growth. Adminis-
tration of  mg Org  resulted in ovulation
induction in .% of patients, as determined by
transvaginal ultrasound and a midluteal rise in pro-
gesterone ().
Org  has activity at the FSHR, in addition to
the LHR, and this lack of selectivity could be an issue
for clinical use. However, it has been shown that ligand
dimerization can aid in subtype selectivity for struc-
turally similar GPCRs. Dimeric compounds, in which
two Org  moieties were joined with flexible
ethylene glycol linkers, or rigid benzene core spacers
have been synthesized and evaluated (). Both series
of compounds had increased LHR selectivity (two- to
fivefold) compared with Org  alone in a lucif-
erase reporter gene assay, either as a result of reduced
potency (rigid linkers) or reduced efficacy (flexible
linkers) at the FSHR ().
Pyrazoles
In , screening of a compound library by a group at
Merck Serono identified pyrazoles as another class of
LMW compounds with activity at the LHR (). From
the results of the initial screen, a focused pyrazole li-
brary was developed, and the activity of compounds
within the library was assessed using a cAMP-specific
scintillation proximity assay in CHO cells stably
expressing human LHR. A compound termed “com-
pound ” (Table ) displayed the best potency (EC
of  nM) and efficacy (% of LH response) ().
Compound  was able to stimulate testosterone
production in cultured rat Leydig cells and elicit a
robust increase in serum testosterone in testosterone-
suppressed -month-old male rats treated with  mg/
kg compound  IP (). No oral bioavailability or
bioactivity of this compound has yet been reported.
Interestingly, compound  was not able to compete
with hCG in LHR radioligand binding assays, sug-
gesting that it also interacts with the receptor at an
allosteric site (). A structure-activity relationship
study determined the allosteric pocket to be large and
lipophilic (similar to the THQ series), as polar groups
were not tolerated as substituents of the pyrazole ring
nitrogen, providing further evidence of localization of
the allosteric site within the receptor TM domain ().
LHR antagonists
Terphenyls
In an effort to identify other allosteric LHR modu-
lators, radiolabeled Org  was used to screen for
novel LMW ligands in a kinetic radioligand binding
assay. A number of hit compounds were identified that
increased the dissociation rate of [H]Org ,
indicative of allosteric modulation of [H]Org 
930 Anderson et al Peptide and Nonpeptide Gonadotropin Analogs
binding (, ). Several analogs were synthesized,
including the potent allosteric inhibitor, compound 
(LUF; Table ). In a luciferase reporter assay, this
compound reduced the potency of LH or Org 
stimulation of the receptor by approximately threefold
().
Analogs with dual-receptor activity
The high degree of sequence conservation among the
glycoprotein hormone receptors frequently translates
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as a loss of selectivity during glycoprotein hormone
receptor LMW compound development. One strategy
used to circumvent this is the creation of bivalent
ligands combining combinations of LMW agonists
and antagonists via linkers to increase specificity of
responses (–). In a study that combined an Org
-based LMW LHR agonist (which also has FSHR
activity) with a very selective LMW FSHR antagonist
based on the THQ compound  (Table ) using an
ethylene glycol spacer, a promising series of analogs
was developed, which exhibited activity at the LHR
(albeit with reduced potency) but no detectable FSHR
activity (). The in vivo activities of these bivalent
compounds have yet to be examined.
Conclusions and Future Perspectives
Gonadotropins from natural and recombinant sources
have been the mainstay of infertility treatment in men
and women for over eight decades and remain a
crucial component of induction of ovulation for
assisted reproduction, thus satisfying the needs of
patients with infertility worldwide. Nevertheless, there
remain considerable challenges to develop less invasive
approaches to minimize multiple injection protocols,
reduce the risk of OHSS, and improve outcomes for
conditions, such as PCOS.
The development of an impressive suite of mol-
ecules, including fused b-subunits, hybrid LH-FSH
molecules, differentially glycosylated gonadotropins,
polypeptide LH and FSH antagonists, longer-acting
analogs, and sustained release formulations has con-
siderable potential, but their full development and use
have yet to be thoroughly explored. A contributing
factor to this is the acquisition of the companies
developing these innovative drugs by large pharma
and subsequent abandonment of the development
programs. Despite this huge endeavor and creativity
in producing a wide range of novel polypeptide go-
nadotropin analogs, the only commercial products
arising from the gonadotropin analog developments
are recombinant gonadotropins and corifollitropin alfa.
Moreover, these recombinant FSH and LH prepara-
tions can still be subject to differential glycosylation with
resulting variation in biological activity. Whereas the
recombinant gonadotropins have advantages of greater
batch-to-batch consistency and purity compared with
Endocrine Reviews, December 2018, 39(6):911–937

hMG and hCG, the latter are still widely used, as they are
less expensive and effective.
Good progress has been made in development of
LMW orally active LHR and FSHR agonists and
antagonists, and three have been evaluated in clinical
studies. The allosteric LHR agonists Org  and
Org  displayed excellent bioavailability, safety,
and pharmacokinetic and pharmacodynamic prop-
erties, and both demonstrated good efficacy in in-
duction of ovulation protocols. Therefore, these
compounds appear to fulfill the major criteria required
for LMW gonadotropin analogs (e.g., specificity, oral
bioavailability, good efficacy). However, evolving
priorities of the pharmaceutical companies running
these programs have meant that further clinical de-
velopment of these compounds has been halted.
As multiple injections of FSH are required for
oocyte recruitment, LMW orally active FSH agonists
would be desirable substitutes. An LMW FSHR al-
losteric agonist (MK-; MSD), developed from a
class of compounds that had promising activity in rat
models, had no effect on follicular development or
estradiol production but an effect on thyroid function
(). Thus, further research is required to produce
efficacious orally active FSHR LMW agonists as
substitutes for multiple injections of conventional
polypeptide FSH with high specificity.
The observation that MK- has thyroid activity
serves to highlight the importance of good-selectivity
margins for these LMW compounds, which has been a
limiting factor for the development of many of these
analogs. Although peptide analog preparations with
mixed LHR/FSHR activity have been/are used clini-
cally (e.g., hMG), “pure” analogs with activity at only
one receptor are preferred, as this enables design of
personalized treatment regimens. In addition, differing
potencies of “mixed activity” LMW analogs at the
different receptors may have implications for the
design/efficacy of clinical protocols.
As mentioned previously regarding polypeptide
gonadotropin molecules, another major stumbling
block to the realization of these exciting possibilities
for novel therapeutics has been the acquisition of
reproductive health companies by big pharma and the
termination of gonadotropin analog development in
view of different priorities of the acquiring companies.
The acquisitions of Organon and Schering halted the
promising LHR allosteric agonist program. Likewise,
the acquisition of Wyeth by Pfizer may have halted
development of the FSHR agonist. Encouragingly, a
number of small biotech companies (e.g., Addex
Therapeutics and TocopheRx) have taken up the
baton, and we may yet see LMW analogs enter the
clinic for a diversity of applications.
One exciting developing area of drug discovery is in
the arena of allosteric modulators, which can modu-
late the effects of exogenous or endogenous ligands,
either positively (PAMs) or negatively (NAMs). These
doi: 10.1210/er.2018-00052
REVIEW
compounds offer subtle advantages over traditional
agonist and antagonist ligands, such as improved
specificity and thus, reduced side-effects. Therefore,
development of PAMs/NAMs that target the go-
nadotropin receptors would be an exciting develop-
ment in this field and is one currently being pursued
by companies, such as Addex Therapeutics.
Another exciting development in the arena of
LMW gonadotropin analogs is their ability to restore
function to inactivating mutations in human gonad-
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otropin receptors. Not only have LMW LHR allosteric
agonists been shown to act as pharmacological chap-
erones and restore cell-surface expression of intracellu-
larly retained mutant LHRs () (an outcome of
the majority of inactivating point mutations in the
LHR) (), but also, function of binding-deficient and
signaling-deficient mutants can be restored (). As
there is no current treatment for these patients, the small
molecules potentially offer a breakthrough in personal-
ized and precision medicine in reproduction, with
broader implications for treating a wide spectrum of
diseases arising from inactivating mutations in GPCRs.
In addition to the application of gonadotropin
analogs as regulators of reproduction, agonists and
antagonists of upstream regulators of GnRH (kiss-
peptin and neurokinin B) are emerging as subtle and
precise regulators of gonadotropin secretion (–). “Future roles and novel
applications of gonadotropins
These show promise of more closely simulating nor- targeting extragonadal tissues
mal physiology as a result of their more proximal are emerging.”
position in the axis regulating GnRH. For example,
kisspeptin has been shown to be effective in a more
gentle induction of ovulation, which may reduce the
risk of OHSS (). Therefore, these upstream regu-
lators may have substantial application as surrogates
for gonadotropin therapies. The near-normal FSH and
low LH in patients with inactivating mutations of
neurokinin B or its receptor () suggested that
neurokinin B antagonists may be able to reverse the
high LH-to-FSH ratio, characteristic of PCOS, and
this has now been demonstrated (). As hot flashes
coincide with LH pulses, the slowing of LH pulses with
neurokinin B antagonists would potentially ame-
liorate the condition, which has has now been
shown (–).
Future roles and novel applications of gonado-
tropins targeting extragonadal tissues are emerging
and may herald a new era of gonadotropin thera-
peutics. Postmenopausal women have low estrogens,
elevated FSH, concomitant bone loss, and increased
body fat, suggesting that FSH, as well as low estrogens,
may be playing a role. In an ovariectomized mouse
model, an antibody to FSHb was found to robustly
inhibit bone resorption and stimulate bone synthesis
(). In another study, the antibody was shown to
induce thermogenic adipose tissue and reduce body fat
(). There is now substantial literature on the
subject, which is beyond the scope of this review. The
reader is therefore referred to comprehensive reviews
https://academic.oup.com/edrv 931
REVIEW

on this subject (, ). Despite an increasing body
of evidence supporting a role for FSH in bone and fat
regulation, there are contradictory findings in both
human and rodent studies, and robust conclusions are
difficult to tease out in view of the relation of estrogen
to FSH and the often-opposed effects of the two
hormones, such as on osteoclasts ().
Elevated LH may also exert neurodegenerative
effects, such as those seen in Alzheimer’s disease
(, ). Additionally, increased FSH and FSHR ex-
for FSH/FSHR in ovarian cancer (–) open up an
even wider spectrum of potential novel applications for
LH and FSH agonists and antagonists.
Although these observations are compelling, the
extent of gonadotropin receptor expression in extra-
gonadal tissues and the effects of gonadotropins on
diverse tissues remain to be fully explored, using a
range of experimental approaches and advanced ep-
idemiological studies. Therefore, it remains to be seen
whether gonadotropin analogs may have use in these

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pression in prostatic cancers and a controversial role applications.

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Acknowledgments
Financial Support: The research of R.C.A., C.L.N., and
R.P.M. from the Centre for Neuroendocrinology is supported
by grants from the University of Pretoria, South Africa;
National Research Foundation (NRF), South Africa; Tech-
nology Innovation Agency (TIA), South Africa; and National
Health Laboratory Services (NHLS) Trust, South Africa. The
research of R.A.A. is supported by a grant from the Medical
Research Council (MRC), United Kingdom.
https://academic.oup.com/edrv
REVIEW
Current Affiliation: R. P. Millar and C. L. Newton’s
current affiliation is Biomedical Sciences, College of Medicine
and Veterinary Medicine, University of Edinburgh, Edinburgh
EH8 9XD, United Kingdom.
Correspondence and Reprint Requests: Robert P.
Millar, PhD, Centre for Neuroendocrinology and Department
of Physiology, Faculty of Health Sciences, University of Pre-
toria, P.O. Box 2034, Pretoria 0001, South Africa. E-mail: bob.
millar@up.ac.za.
Disclosure Summary: R.A.A. and R.P.M. undertake
consultancy work for KaNDy Therapeutics Ltd. The
remaining authors have nothing to disclose.
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Abbreviations
7TM, seven transmembrane; aa, amino acid; ART, assisted
reproductive technology; CHO, Chinese hamster ovary; CTP,
carboxy-terminal peptide; ECD, extracellular N-terminal
domain; ECL, extracellular loop; FcRn, neonatal Fc receptor;
FSHR, FSH receptor; GnRHR, gonadotropin-releasing hor-
mone receptor; GPCR, G protein–coupled receptor; hCG,
human chorionic gonadotropin; hCGba, human chorionic
gonadotropin b-subunit fused to the N-terminus of the
gonadotropin a-subunit; HH, hypogonadotropic hypo-
gonadal; hMG, human menopausal gonadotropin; HPG,
hypothalamic-pituitary-gonadal; LHCGR/LHR, LH receptor;
LMW, low molecular weight; LRR, leucine-rich repeat; MSD,
Merck Sharpe & Dohme; NAM, negative allosteric modu-
lator; OHSS, ovarian hyperstimulation syndrome; PAM,
positive allosteric modulator; PCOS, polycystic ovarian syn-
drome; PEG, polyethylene glycol; PLC, phospholipase C;
PMSG, pregnant mare’s serum gonadotropin; PO, orally; SC,
subcutaneous(ly); THQ, tetrahydroquinoline; TM, trans-
membrane; TSHR, TSH receptor; TZD, thiazolidinone.
937