The Role of LH in Controlled
Ovarian Stimulation

16

Sandro C. Esteves and Carlo Alviggi

Abstract

Although exogenous FSH is the main regulator of follicular growth instimulated cycles, LH plays a key role in promoting steroidogenesis andfollicle development. Stimulation protocols with LH supplementation
are mandatory in patients with hypogonadotropic hypogonadism who
do not achieve adequate steroidogenesis by stimulation with FSH alone,
but resume adequate estrogen production by LH supplementation. In
normogonadotropic women undergoing controlled ovarian stimulation(COS), the hypogonadotropic state after GnRH analogues is short induration, and the resting levels of LH are usually sufﬁcient for promoting optimal follicular development. An increased body of evidence otherwise indicates that at least three subgroups of normogonadotropic
patients indeed seem to beneﬁt from the addition of LH activity to thestimulation protocol: (1) patients >35 years, (2) patients with a decreased
ovarian reserve/poor response to COS (poor responders), and (3)patients with an initial poor response to rec-hFSH (hyporesponders).
Possible reasons for a beneﬁcial effect of LH activity supplementation
include the biological aging of the ovary and pharmacogenetics involving the LH molecule and its receptor. The three gonadotropins containing LH activity are human menopausal gonadotropin (hMG), with 1:1
ratio of FSH/LH in which LH activity is driven by hCG; recombinant

S.C. Esteves, MD, PhD (*)
ANDROFERT, Andrology and Human Reproduction
Clinic, Av. Dr. Heitor Penteado, 1464 Campinas,
São Paulo 13075-460, Brazil
e-mail: s.esteves@androfert.com.br
C. Alviggi, MD, PhD
Department of Neuroscience and Reproductive
Medicine, University of Naples Federico II,
Naples, Italy© Springer India 2015
S. Ghumman (ed.), Principles and Practice of Controlled Ovarian Stimulation in ART,
DOI 10.1007/978-81-322-1686-5_16

s.esteves@androfert.com.br

171

S.C. Esteves and C. Alviggi

172

human LH (rec-LH), with only LH activity driven by pure LH; and a
combination of recombinant FSH and recombinant LH, with 2:1 ratio ofpure FSH/LH activity. In addition to the higher purity and speciﬁc
activity of rec-hLH compared with hMG, LH activity is markedly different at the molecular and functional levels between these gonadotropins. The choice of the type of gonadotropin preparations containing
LH activity should be considered when tailoring COS with LH supplementation because they may inﬂuence cycle outcome.
Keywords

Gonadotropins • Luteinizing hormone • Human chorionic gonadotropin •Controlled ovarian stimulation • Assisted reproductive technology

Abbreviations

rec-hCG

3β-HSD
AA
ART
Asn
cAMP
CHO
CI
COS
DHEA
FbM
FSH
GalNAcGC
GlcNAcGnRH
hCG
hMG
HP-hMG

16.1

3β-Hydroxysteroid dehydrogenase
Amino acids
Assisted reproductive techniques
Asparagine
Cyclic adenosine monophosphate
Chinese hamster ovaryConﬁdence interval
Controlled ovarian stimulation
Dehydroepiandrosterone
Filled by mass
Follicle-stimulation hormone
N-acetylgalactosamine
Granulosa cells
N-acetyl glucosamine
Gonadotropin-releasing hormone
Human chorionic gonadotropin
Human menopausal gonadotropin
Highly puriﬁed human menopausal
gonadotropin
ICSI
Intracytoplasmic sperm injection
IGF
Insulin-like growth factor
IVF
In vitro fertilization
LH
Luteinizing hormone
LMW
Low molecular weightOR
Odds ratio
P4
Progesterone
P450arom P450 aromatase
P450scc
Cholesterol side-chain cleavage
enzyme
RCT
Randomized controlled trial
RD
Risk difference

Recombinant human chorionicgonadotropin
rec-hFSH
Recombinant human folliclestimulating hormone
rec-hLH
Recombinant human luteinizing hormone
RR
Relative risk
SDS-PAGE
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
SE-HPLC
Size-exclusion high-performanceliquid chromatography
SO3-4GalNAc Sulfonated
β1–4-linked
N-acetylgalactosamine
StAR
Steroidogenic acute regulatory
protein
WMD
Weighted mean difference

Introduction

Gonadotropin therapy has a central role in ovarian stimulation. Its introduction in medical practice dates from almost one century ago andrepresents a major upgrade in infertility treatments. Treatment of anovulatory women withexogenous gonadotropin administration started
in the 1960s and expanded to ovulatory women to
promote multifollicular development in the 1980s[1–3]. Gonadotropins were ﬁrst extracted fromurine in the 1940s, and a decade later the ﬁrst urinary forms of human chorionic gonadotropin(hCG) and human menopausal gonadotropins.esteves@androfert.com.br

16

The Role of LH in Controlled Ovarian Stimulation(hMG) became commercially available [2, 3].
Improvements in the puriﬁcation methods led tothe production of follicle-stimulating hormone(FSH) – only products in the 1980s and advances
in DNA technology enabled the development ofrecombinant human gonadotropins, whichbecame commercially available approximately
two decades later [2–4]. In 2000, recombinanthuman luteinizing hormone (rec-hLH) becamecommercially available, and recently, in 2007, aﬁxed combination of recombinant FSH (rechFSH) and rec-hLH was launched [3].
Although exogenous FSH is the main regulator of follicular growth in stimulated cycles, thequestion whether the LH hormonal environment
achieved after administration of gonadotropinreleasing hormone (GnRH) analogues is really
optimal for all categories of patients undergoing
controlled ovarian stimulation (COS) or whether
subgroups of patients exist that might actually
beneﬁt from exogenous LH supplementation hasreceived increased attention. While FSH is themain antral follicular growth regulator, LH plays
a key role in promoting steroidogenesis anddevelopment of the leading follicle and has different functions in different stages of both naturaland stimulated cycles. During the early follicularphase, LH stimulates the production of androgens by theca cells. Androgens are then transferred to the GC and transformed into estrogens
via aromatization [4]. From the mid-follicularphase onwards, LH upregulates FSH receptor
expression and sustains FSH-dependent granulosa cell activities, including aromatase production and growth factors’ release. In addition, LH
sustains follicular growth and ﬁnal follicularmaturation via its direct effects on the GC in thelate follicular phase [4]. Therefore, during recent
years an increasing body of evidence has emerged
examining the possible beneﬁcial role of exogenous LH activity supplementation in stimulated
ART cycles.
The purposes of this chapter are (1) to review
the glycoprotein structure and action of luteinizing hormone (LH), (2) to examine the rationaleof using luteinizing hormone (LH) supplementation during controlled ovarian stimulation, (3)to present the clinical evidence supporting LH

173

supplementation during COS in different subset
of infertility patients, (4) to describe the commercially available preparations containing LH
activity, and lastly (5) to analyze the differencesin LH activity provided by rec-hLH and hMG
preparations.

16.2

Structure and Function of LH

LH is a protein covalently linked to a carbohydrate (glycoprotein). It is synthesized andsecreted by gonadotrophs of the anterior pituitary
gland under stimulation of the pulsatile secretionof gonadotropin-releasing hormone (GnRH)
from the hypothalamus [5]. The LH moleculecomprises two non-covalently linked protein subunits, alpha and beta. The three-dimensional
structure and the active conformation of the subunits are maintained by internal disulﬁde bonds[6]. The alpha subunit contains 92 amino acids(AA) and is identical in all gonadotropins (i.e.,
LH, FSH, and hCG). The beta subunit differs in
the aforementioned gonadotropins and confers
unique receptor speciﬁcity as well as differential
biological and immunological properties(Fig. 16.1) [7]. Protein subunits alone have nobiologic activity; the latter is provided by glycosylation, which is achieved by the attachment ofcarbohydrate moieties forming heterodimers [3].
The extent and pattern of glycosylation convey
the differential spectrum of charges, bioactivities,
and half-lives of each gonadotropin [7, 8]. The
LH molecule is further modiﬁed in vivo by theaddition of a sialic acid (sialylation) or sulfonicgroup (sulfonation) to the carbohydrate moieties.
Both sialylation and sulfonation are physiological processes with major roles in gonadotropin
biological activity modulation [3, 7–9].
Elimination of LH from circulation is modulatedby the number of glycosylation sites and sialic
acid residues attached to the carbohydrate moieties [10]. LH beta subunits contain a single siteof N-linked glycosylation (Asn 30) and few sialic
acid residues (only 1 or 2); as such, native LH hasa short half-life of only 20–30 min (Fig. 16.2) [8,10]. LH shows physiologic ﬂuctuations in isoform proﬁle during the menstrual cycle. Mores.esteves@androfert.com.brS.C. Esteves and C. Alviggi

174

Fig. 16.1 Luteinizing hormone and human chorionicgonadotropin molecules. (a) LH is a glycoprotein with
two subunits, the alpha subunit (red), similar to that of
FSH and hCG with two carbohydrate attachment sites,
and the beta subunit (blue), with only one carbohydrate
attachment site. The light blue balls represent the carbohydrate chains. (b) hCG is similar in its structural

attributes to LH. A notable exception is the presence of along carboxyl terminal segment that is O-glycosylated(O-linked CHO), conferring longer half-life to hCG. The
alpha and beta subunits are represented in red and blue
strands, respectively, whereas the light blue balls represent the carbohydrate chains (Adapted from Leão andEsteves [3])Fig. 16.2 Glycosylation
patterns of LH and hCG. The
alpha subunits of eachhormone are identical inamino acid sequence andcontain two sites of N-linked
glycosylation. The beta
subunit confers hormone
speciﬁcity and contains
variable amounts of N-linked
glycosylation. LH beta
subunit contains a single siteof N-linked glycosylation,
while hCG beta subunit
contains two sites ofN-linked glycosylation. In
addition, hCG has anextended C-terminal that
contains four sites ofO-linked glycosylations.esteves@androfert.com.br

16

The Role of LH in Controlled Ovarian Stimulation

175

Fig. 16.3 LH/hCG receptor.
LH receptors are included in
the subgroup of G proteincoupled receptors. Such
receptors are characterizedby having 7-transmembrane
domains and a large
N-terminal extracellularregion, which is the
predominant site of hormone
binding

basic LH isoforms are seen at midcycle due toconsiderably decreased sulfonation concomitantwith slightly increased sialylation. Both changes
increase LH half-life in the circulation, thus
explaining the increased levels of serum LH at
this period. This change in isoform proﬁle seems
to be physiologically important for ovulation
triggering [11].
LH binds to a subgroup of G protein-coupled
receptors with 7 transmembrane domains and alarge N-terminal extracellular region (Fig. 16.3)[12, 13]. Receptor activation requires that hormones bind to the N-terminal region, thus leading to intramolecular signal transduction fromthe ligand–receptor complex to the transmembrane domains. Although the mechanism that
underlies this intramolecular signaling pathway
is not fully understood, it involves stimulation of
adenyl cyclase via coupling to Gs proteins [12,13]. Unlike FSH receptors that are expressed
exclusively in the granulosa cells (GC), LH
receptors are expressed in both GC and thecacells. The LH receptor expression is at its maximum in the GC of preovulatory follicles, but
antral follicles with 3–10 mm in diameter havealready expressed these receptors at approximately 10 % of the maximum [14].

16.2.1 The Role of LH on Ovarian
Steroidogenesis
The two-cell system, ﬁrst proposed by Falck in
1959, is based on the assumption that while FSH
receptors are present only in the GC, LH receptors
are present in the theca cells and absent in the GC
during the early follicular stages [15–18]. Thecacells are characterized by exhibiting steroidogenic
activity in response to LH stimulation. Speciﬁcally,cholesterol is converted into androgens (i.e., testosterone and androstenedione) by transcription activities of cholesterol side-chain cleavage enzyme(P450scc), P450c17, and 3β-hydroxysteroid dehydrogenase (3β-HSD) genes. The starting point ofsteroid biosynthesis is cholesterol, a carbon 27(C27) steroid. Cholesterol is converted to pregnenolone (C21) by P450scc (CYP11A – cytochrome
P450, family 11, subfamily A, polypeptide 1),
whose regulation is mediated by steroidogenic
acute regulatory protein (StAR). StAR facilitates
the inﬂux of cholesterol into the mitochondria
where P450scc is located. StAR expression isenhanced by cAMP and by stimulation of GC with
FSH and LH or hCG [3, 4, 18, 19].
The primary route of pregnenolone metabolism
is via the delta 5 pathway, the ﬁrst two steps of whichs.esteves@androfert.com.brS.C. Esteves and C. Alviggi

176

Theca cells

11
1 19
2

21 22 2420
12 18 17 23
13 16
14

HO 3 4 5

6

Granulosa cells

Δ4pathway

26
25
27

21 22 2420
12 18 17 23
11
13 16
1 19
2
14
15
8
7
HO 3 4 5 6Cholesterol

1587Cholesterol

stAR CYP11A

CH3

CH3

(P450scc)

Δ4
pathway

27

stAR CYP11A

C=O

C=O

26
25

CH3

CH

3

(P450scc)

C=O

C=O

O
Pregnenolone

HO

Progesterone

Pregnenolone

CYP17 (P45017a hydroxylase)Δ5
pathwayCH
CH

O
Progesterone

3

C=O
3

C=O

3β-HSD

HO

OH

OH

3β-HSDHOO
17-Hydroxyprogesterone

17-Hydroxypregnenolone

CYP17 (P45017a lyase)
O

O

O

O

HO
DHEA

AndrostenedioneOH

CYP19 (aromatase)17β-HSD

Estrone (E1)HO

17b- HSD
OH

OH
Estradiol -17b (E2)

O
HO

HO
Androstenediol

Testosterone

StAR = steroidogenic acute regulatory protein
3β- HSD = 3β - hydroxysteroid dehydrogenase17β- HSD = 17β - hydroxysteroid dehydrogenase

Fig. 16.4 Human ovarian steroidogenesis. The starting
point for steroid biosynthesis is the conversion of cholesterol in pregnenolone by P450scc. One route of pregnenolone metabolism is the delta-5 pathway (red arrows) by
the action of CYP17 (P450c17). Hydroxylation of pregnenolone
at
the
C17a
positionforms
17-hydroxypregnenolone, and subsequent removal of theacetyl group forms the androgen precursor dehydroepiandrosterone (DHEA). Another route of pregnenolone

metabolism is the delta-4 pathway (purple arrows) inwhich pregnenolone is converted to progesterone by theaction of 3b-HSD (an irreversible conversion).
Progesterone is then converted to 17-hydroxyprogesterone
by CYP17. In humans, 17-hydroxyprogesterone cannot
be further metabolized. Aromatization of androgens to
estrogens is a distinct activity within the granulosa layer
induced by FSH via activation of the P450 aromatase(P450arom) gene (From Leão and Esteves [3])are driven by the same enzyme, CYP17 (P450c17).
The hydroxylation of pregnenolone at the C17aposition forms 17-hydroxypregnenolone, and thesubsequent removal of the acetyl group forms
the androgen precursor dehydroepiandrosterone(DHEA). Accordingly, CYP17 has both hydroxylase and lyase activity. Lastly, DHEA is converted

to androstenedione by 3β-HSD [15, 19, 20]. A secondary route of metabolism involves the conversion of pregnenolone to progesterone by the actionof 3β-HSD via the delta 4 pathway. Progesterone
is then converted to 17-hydroxyprogesterone by
CYP17 (Fig. 16.4) [20]. Importantly, CYP17 is
located exclusively in thecal and interstitial cells, thes.esteves@androfert.com.br

16

The Role of LH in Controlled Ovarian Stimulation

177

Fig. 16.5 The “two-cell” system. FSH receptors are present exclusively in the granulosa cells. LH receptors arepresent in the theca cells and initially absent in the granulosa cells. In response to LH, theca cells convert cholesterol to androgens (testosterone and androstenedione).
CYP17 is located exclusively in thecal cells, whereas
CYP19 (aromatase) is expressed only in the granulosa.

Thus, androgens must diffuse into the granulosa layer tobe converted to estrogen via aromatization induced by
FSH. Both FSH and LH act via AMPc production. In thelate follicular phase, FSH induces LH receptor formationin the granulosa cells, which acquire LH responsiveness.
In the granulosa, LH enhances FSH action (increasing
estrogen production)

extrafollicular compartment of the ovary, whereas
CYP19 (aromatase), that converts androgens to
estrogens, is expressed exclusively in GC, the intrafollicular compartment [20–22]. Thus, aromatization of androgens to estrogens is a distinct activity
within the granulosa layer induced by FSH via
activation of the P450 aromatase (P450arom) gene.
Androgens produced in the theca layer must therefore diffuse into the granulosa layer to be converted
to estrogens (Fig. 16.5). Hence, increasing levelsof estradiol in the peripheral circulation during thefollicular phase reﬂect the release of estrogen from
granulosa cells into blood vessels [15, 22]. Thecaand granulosa cells also secrete peptides that act asboth autocrine and paracrine factors. Insulin-likegrowth factor (IGF) is secreted by theca cells andenhances LH-mediated androgen production within
the thecal compartment as well as FSH-mediatedaromatization in granulosa cells [15]. Inhibin
and activin are produced in the granulosa cells in
response to FSH and modulate the expression ofsteroidogenic enzymes, especially P450c17 in
theca cells. While inhibin enhances androgen synthesis, activin has an opposite effect. Activin also
has the important autocrine role of enhancing FSH
action mainly by increasing the expression of FSH
receptors [15] (Fig. 16.6).

LH also acts in GC to stimulate progesterone
production. Most circulating progesterone (~95 %)is produced in the intrafollicular compartment by
the granulosa cells via the action of 3b-HSD thatcatalyzes conversion of pregnenolone (delta-4
pathway) under the LH inﬂuence (see Fig. 16.4)[18, 22]. Despite a marked increase in progesterone levels measured at the veins of the active
ovary in the mid-follicular phase, peripheral concentrations increase only slightly probably due
to active liver metabolism [23]. Progesterone canbe further converted to 17-hydroxyprogesterone
by CYP17 (via delta-4 pathway). However, very
little 17-hydroxyprogesterone is converted toandrostenedione, since human CYP17 catalyzes
this reaction at only 3 % of the rate for the conversion of 17-hydroxypregnenolone to DHEA [18,22, 24]. Therefore, 17-hydroxyprogesterone isbasically the ﬁnal product of the delta-4 pathway
in humans. Moreover, progesterone itself cannot
be metabolized in the GCs because CYP17 is not
expressed within this cell compartment; as such,progesterone is the ﬁnal product of the delta-4
pathway in the intrafollicular compartment andcannot be converted to estradiol in the GC underthe effect of LH [20]. The preovulatory rise in progesterone facilitates the positive feedback action ofs.esteves@androfert.com.brS.C. Esteves and C. Alviggi

178

Fig. 16.6 Modulation of steroidogenic enzymes. In theearly follicular phase, inhibin and activin are produced in
the granulosa cells in response to FSH. They have important paracrine functions to modulate the expression of steroidogenic enzymes, especially P450c17 in theca cells.
Inhibin enhances LH function, thus stimulating androgensynthesis to latter aromatization to estrogen in the granulosa, whereas activin suppresses androgen synthesis.
Activin has also an important autocrine role of enhancing
FSH action, especially by increasing the production of
FSH receptors. Production of inhibin by the granulosa

cells is increased in the late follicular phase while activin
is decreased, with a positive effect on androgen production by theca cells. FSH induces LH receptor formation in
the granulosa cells, which acquire LH responsiveness andtherefore less FSH dependence. In granulosa, LH
enhances FSH action that in turn increases estrogen production, initiates progesterone production (negatively
modulated by activin), and control granulosa productionof inhibin. The increase in inhibin, in turn, suppresses
FSH secretion by the pituitary, important to ensure thedominance of the follicle

estrogen on the pituitary; the latter is the key factor to induce the midcycle LH peak in the natural
cycle. Progesterone also stimulates a midcycle
FSH surge, important to support the full expressionof LH receptors at the granulosa layer [22, 25].
In summary, ovarian steroidogenesis is theresult of combined LH and FSH stimulation of
the two cell types, theca and granulosa, inﬂuenced by autocrine and paracrine factors.

its receptors activates cyclic AMP-protein kinase
A (cAMP/PKA) pathway, which represents anadditional stimulus to follicular growth [27].
Thereby, the maturing follicle also reduces its
dependency on FSH by acquiring LH receptors
and LH responsiveness [26–30]. FSH and LH
cooperate in inducing the local production of
the soluble molecule inhibin B and growth factors. Among these, insulin growth factors (IGF)
I and II, which are expressed by both granulosa
and theca cells throughout folliculogenesis, areimportant in promoting follicular maturation [31, 32].
Furthermore, LH exerts an antiapoptotic effect on
the GCs, mediated by the production of ﬁbroblast
growth factors that maintain calcium homeostasis
and granulosa cell viability by stimulating calcium efﬂux via a protein kinase C (PKC) deltadependent pathway [33]. Additional signaling

16.2.2 The Role of LH on Follicular
Maturation and Luteal Phase
SupportIn the mid-follicular phase, FSH induces LH
receptor expression in the granulosa cells ofdeveloping follicles [26]. The action of LH on

s.esteves@androfert.com.br

16

The Role of LH in Controlled Ovarian Stimulation

pathways (e.g., AKT and ERK1/2 pathways)
involve the expression of EGF-like growth factorsthat inﬂuence GC proliferation, differentiation,and survival (apoptosis blockage) [34, 35]. Lastly,
aromatase expression and steroidogenic function
via LH receptor activation are likely to involvecAMP/PKA,
extracellularsignal-regulated(ERK) 1 and 2, and AKT pathways, all playing
a crucial role in the ﬁnal stages of maturation ofhuman oocytes and follicles [36, 37].
LH activity during the luteal phase is totally
responsible for the maintenance and the steroidogenic activity of the corpus luteum [38]. LH is
responsible for the upregulation of growth factors
like vascular endothelial growth factor A [39,40], which plays a dynamic role in luteal angiogenesis, and epidermal growth factor-like ligands,
amphiregulin and epiregulin, which regulate
apoptosis in luteinized human granulosa cells[34, 41–44]. Furthermore, LH stimulates expression of extragonadal LH receptors in the endometrium [45, 46] and production of cytokines
involved in implantation [47].
Therefore, LH regulates both granulosa andtheca cells and has a pivotal role in folliculardevelopment and maturation. In light of theaforementioned ﬁndings, we can conclude that
(1) both gonadotropins contribute (via granulosa)to maintain the autocrine–paracrine system governing dominant follicle’s growth; (2) LH is crucial in sustaining FSH activity in the granulosa
during intermediate–late stages of folliculogenesis; and (3) LH is critical for maintaining corpus
luteum function during the luteal phase.

16.3

Rationale of LH
Supplementationin Stimulated Cycles

The “LH window” concept, as outlined by
Shoham in 2002, proposes that in the absence ofa threshold level of serum LH, estradiol production will be insufﬁcient for follicular development, endometrial proliferation, and corpus
luteum formation [48]. This concept can beclearly observed in patients with hypogonadotrophic hypogonadism who do not achieve adequate

179

steroidogenesis by stimulation with FSH alone,
but resume sufﬁcient estradiol production by LH
supplementation [49]. Evidence therefore suggests that in reproductive cycles optimal follicular development occurs within a “LH window,”that is, above an LH threshold of 1.1 and below
an LH ceiling of 5.1 IU/L [48, 49].
After pituitary suppression, still widely usedin association with COS, residual circulating levels of endogenous LH are usually adequate tosupport multiple follicular growth and oocyte
development in COS with gonadotropins devoidof LH activity [50, 51]. In fact, only 1 % of LH
receptors need to be occupied to drive adequate
ovarian steroidogenesis. Fair evidence indicatesthat most normogonadotropic women have sufﬁcient levels of endogenous LH and do not require
exogenous LH supplementation [52–54]. Despiteof that, a recent large meta-analysis including a
total of 40 RCTs and 6443 women aged 18–45
years found a small relative increase (estimate of
9 %) in clinical pregnancy rate in patients treatedwith of rec-hFSH plus rec-hLH versus rec-hFSH
alone (RR 1.09; 95 % CI 1.01–1.18) [55]. More
importantly, ovarian response to COS with FSHonly-containing gonadotropins has shown to be
suboptimal in subsets of normogonadotropic
women, including those with advanced reproductive age (≥35 years old) [56, 57], diminishedovarian reserve [54, 58], and highly suppressed
levels of endogenous LH, in whom LH activity
falls below the LH threshold [59–63]. In addition, a subgroup of normogonadotropic patients
who had normal estimated ovarian reserve but
suboptimal responses to FSH-alone stimulation
has also been identiﬁed and termed “hyporesponders” [64–67].
Clinical evidence indicates that the aforementioned subgroups have less responsive ovaries instimulated cycles with FSH, which could be
explained by a wide range of factors, including
reduced paracrine ovarian activity [68], genetically determined reduced LH bioactivity [69],
reduced androgen secretory capacity [70], anddecreased number of functional LH receptors[71]. Serum androgen levels, especially total testosterone (T), calculated free T, dehydroepiandrosterone sulfate, and androstenedione, declines.esteves@androfert.com.brS.C. Esteves and C. Alviggi

180

steeply with age, with the decline of each being
greater in the early reproductive years than thelater decades [72, 73]. Hence, it has been hypothesized that such women would beneﬁt from
LH-containing
gonadotropin
preparations.
Action of LH at the follicular level could promote
an increase in ovarian steroidogenesis and androgen production for its later aromatization into
estrogens, with a positive impact on the follicularmilieu. Furthermore, LH has also a direct effecton follicular growth and maturation via different
signaling pathways that positively impact oocyte
quality [74].

16.4

Clinical Evidence Supporting
LH Supplementation During
COS in Selected Patients

16.4.1 Hypogonadotropic
Hypogonadism
The European Recombinant Human LH Study
Group investigated the efﬁcacy of rec-hLH forsupporting FSH-induced follicular development
in hypogonadotropic hypogonadal women (LHlevels of <1.2 IU/l; WHO group I anovulation)[49]. Thirty-eight patients were randomized toreceive 0, 25, 75, or 225 IU/day of rec-hLH in
addition to a ﬁxed dose of rec-hFSH (150 IU/day).
The authors found that rec-hLH was able to promote a dose-related increase in estradiol andandrostenedione secretion by rec-hFSH-induced
follicles. Serum concentrations on the last day
of FSH administration were 65 ± 4, 195 ± 94,
1392 ± 585, and 2441 ± 904 pmol/L for E2 and3.6 ± 0.9, 5.1 ± 1.3, 6.4 ± 1.3, and 6.7 ± 1.3 nmol/L
for androstenedione in the patients treated with
0, 25, 75, and 225 IU rec-hLH, respectively. LH
supplementation also increased ovarian sensitivity to FSH, as shown by the proportion of patients
who developed follicles after the administrationof a deﬁned dosage of FSH. While only 12.5 %of the patients treated with FSH alone developedfollicles, the proportion substantially increased,according to the varying doses of rec-hLH(42.8 % in 25 IU and 77.8 % and 80 % in 75 IU
and 225 IU, respectively). Furthermore, follicles

that had been exposed to rec-hLH showed anincreased ability to luteinize after hCG exposure.
In the aforementioned study, a daily dose of
75 IU rec-hLH was effective in the majority ofwomen in promoting optimal follicular development (deﬁned as > or = 1 follicle > or = 17 mm;
E2, > or = 400 pmol/L; midluteal phase progesterone, > or = 25 nmol/L) and maximal endometrial growth. Lastly, rec-hLH was shown not tobe immunogenic and was well tolerated by thepatients.
In conclusion, exogenous LH activity supplementation is mandatory in stimulation protocols applied to women with hypogonadotropic
hypogonadism.

16.4.2 Older Women (>35 Years Old)
The impact of luteinizing hormone administration in ovarian stimulation with gonadotropinreleasing hormone (GnRH) antagonist cycles was
examined by Bosch and colleagues in a randomized controlled trial (RCT) involving 720 women
undergoing their ﬁrst or second IVF [75]. The
authors compared cycle outcome, according to
the use of rec-hFSH or rec-hFSH + rec-hLH in anage-adjusted analysis. For the patients <36 years
old, the total starting dose of gonadotropins was
225 IU/d for both stimulation protocols. In the
rec-hFSH-alone group, 225 IU/d SC of rec-hFSH
was administered, and the starting dose for the
rec-hFSH + rec-hLH group was 150 IU/d of rechFSH and 75 IU/day of rec-hLH. As noted, LH
supplementation was started on stimulation day
1. For those patients aged 36–39 years, the total
starting dose of gonadotropins was 300 IU/d forboth study groups: rec-hFSH-alone, 300 IU/dSC of rec-hFSH was administered, and for the
rec-hFSH + rec-hLH group, 225 IU/d of rechFSH + 75 IU/d rec-hLH. The rec-hLH dose
remained ﬁxed across the cycle. In the younger
population (up to 35 years old), implantation rates were similar, 27.8 % versus 28.6 %,odds ratio (OR) 1.03 (95 % conﬁdence interval[CI] 0.73–1.47), as was the ongoing pregnancy
rate per started cycle, 37.4 % versus 37.4 %,OR 1.0 (95 % CI 0.66–1.52). In older patientss.esteves@androfert.com.br

16

The Role of LH in Controlled Ovarian Stimulation(36–39 yrs.), the implantation rate was signiﬁcantly higher in the rec-hFSH + rec-LH group:
26.7 % versus 18.6 %, OR 1.56 (95 % CI 1.04–
2.33). Ongoing pregnancy rates per started cycle
were not statistically different: 33.5 % versus
25.3 %, OR 1.49 (95 % CI 0.93–2.38).
Contrary results have been reported by Konig
et al. in an RCT involving 253 couples undergoing IVF/ICSI [76]. In their study, women were 35
years or older and received ovarian stimulation ina GnRH antagonist protocol with either rechFSH 225 IU/day or rec-hFSH + rec-hLH
150 IU/d starting on stimulation day 6. The
intention-to-treat analysis revealed implantation
rates (18.8 % vs. 20.7 %; mean difference −1.9 %,95 % conﬁdence interval [CI] −8.0 to 11.7) andclinical pregnancy rates (28.0 % vs. 29.7 %;mean difference −1.5 %, 95 % CI −9.4 to 12.7).
A systematic review and meta-analysis of thestudies examining the age-related effects of LH
supplementation in COS were conducted by Hilland colleagues [57]. The authors demonstrated
that LH supplementation in women aged
>34 years old undergoing COS with rec-hFSH
was beneﬁcial. Their study included 7 RCTs (902
women) and compared COS using rec-hFSH
alone or in combination with rec-hLH. GnRHagonist downregulation was used in ﬁve trials,
while GnRH antagonist and GnRH-agonist
micro-ﬂare were used in the remaining trials. The
dose and day of starting rec-hLH supplementation varied among trials. In ﬁve of them a ﬁxed
dose of 150 IU rec-hLH, which started either on
the sixth or seventh stimulation day, was used.
One trial used a ﬁxed 2:1 ratio of rec-hFSH andrec-hLH, while another used a ﬁxed dose of
75 IU rec-hLH regardless of the FSH dose; inboth of them LH supplementation was given
from the ﬁrst day of stimulation on. Implantation(OR = 1.36; 95 % CI: 1.05–1.78, I2 = 12 %) andclinical pregnancy rates (OR = 1.37; 95 % CI:
1.03–1.83, I2 = 28 %) were signiﬁcantly higher
for women who received rec-hLH in addition to
rec-hFSH compared with those in whom rechFSH was administered alone.
The meta-analysis by Hill et al. was subsequently reexamined by Konig and colleagues,
who replaced the study of Bosch and cols. by

181

their own and concluded that no effect whatsoever could be observed by adding LH to older
patients [77]. Nevertheless, a methodologicalbias could have been produced by replacing the
Bosch and colleagues’ study, which represented
36 % of the weight of all studies pooled in the
aforementioned meta-analysis, by the one of
Konig and cols. because the protocols of COS
differed with regard to the day LH supplementation has started; LH supplementation was started
in the mid-follicular phase in the latter in contrast
to the former, in which rec-LH was administeredsince the ﬁrst day of stimulation. It has been suggested that LH supplementation should be initiated on the beginning day of stimulation to get
total advantage of the LH effects on both thecaand granulosa cells [78].
In conclusion, evidence suggests that rechLH supplementation has a positive effect on
cycle outcome of older women (>35 years
old), particularly when used from the start of
COS. Nevertheless, given the heterogeneity ofthe published data, additional large RCTs examining the impact of rec-hLH supplementationfrom the early phases of COS are needed to draw
a conclusive recommendation about the routine
incorporation of rec-hLH in older women undergoing IVF/ICSI.

16.4.3 Poor Responders
Mochtar et al., evaluating poor responders, havedemonstrated the usefulness of adding rec-hLH to
COS. These authors pooled three RCTs including
310 participants and showed that higher ongoing
pregnancy rates (OR = 1.85; 95 % CI: 1.1–3.11)
were obtained in patients treated with the combination of rec-hFSH and rec-hLH compared with
rec-hFSH alone [54]. In another meta-analysis of
Bosdou et al., which included 7 RCTs and 603
patients classiﬁed as poor responders, differencesin clinical pregnancy were not detected in thegroup of patients receiving LH supplementation[58]. Nevertheless, the deﬁnition criteria for poor
responders were not uniform among the included
studies, and two of the RCTs evaluated slow/
hyporesponders rather than poor responders.

s.esteves@androfert.com.brS.C. Esteves and C. Alviggi

182

The protocols of stimulation also varied as
GnRH antagonists and agonists were applied in
two trials each, and GnRH-agonist short protocol was used in three subjects. The way rec-hLH
supplementation was given also varied as daily
doses of either 75 IU or 150 IU were used, andthe starting day differed or was not traced. RechLH was added to rec-hFSH from the ﬁrst stimulation day in one trial, at stimulation day 7 inthree trials, at day 8 in one trial, and on the dayof the ﬁrst GnRH antagonist injection in another
trial. Although statistical signiﬁcance was notreached, the magnitude of the effect size and thewidth of the 95 % CI regarding the clinical pregnancy rates (RD = +6 %; 95 % CI: −0.3 to +13 %;p = 0.06) suggested a potential clinical beneﬁt of
LH supplementation. Nevertheless, the authorsof the aforesaid meta-analyses did ﬁnd that rechLH supplementation was beneﬁcial in terms oflive birth rates after IVF (RD = +19 %; CI: +1
to +36 %), but their results were derived from asingle RCT.
Lately, a large meta-analysis assessed the outcomes of rec-hFSH plus rec-hLH or rec-hFSH
alone for ovarian stimulation in association with
GnRH analogues during ART [55]. A total of 40
RCTs involving 6443 women aged 18–45 years
were included, of which 14 studies (1129
patients) speciﬁcally investigated poor responders. Poor response (POR) was deﬁned according
to study authors’ criteria and although the studies
were published prior to the European Society of
Human Reproduction and Embryology (ESHRE)
consensus deﬁnition of POR [79], in 10 of the 14
studies reporting POR data, the deﬁnition of POR
employed was aligned with the subsequently
reported ESHRE deﬁnition. According to the
ESHRE consensus, POR is deﬁned by the presence of at least two of the following three features: (1) advanced maternal age (≥40 years) or
any other risk factor for POR, (2) a previous POR(≤3 oocytes with conventional stimulation), and(3) an abnormal ovarian reserve test (antral follicle count [AFC] <5–7; anti-Mullerian hormone[AMH] <0.5–1.1 ng/mL), but two episodes of
POR after maximal COS per se are sufﬁcient todeﬁne a patient as poor responder. Patients ofadvanced age with an abnormal ORT may beclassiﬁed as POR since these features indicate
reduced ovarian reserve and act as a surrogate ofovarian stimulation cycle outcome. In this case,the patients should be deﬁned as “expected poor
responder” [79]. In the aforementioned study byLehert and colleagues, signiﬁcantly more oocytes
were retrieved with rec-hFSH plus rec-hLH versus rec-hFSH alone in poor responders (12 studies, n = 1077; weighted mean difference +0.75
oocytes; 95 % CI 0.14–1.36). Also, signiﬁcantly
higher clinical pregnancy rates were observed inthis patient category with rec-hFSH plus rec-hLH
versus rec-hFSH alone (14 studies, n = 1179; RR
1.30; 95 % CI 1.01–1.67; ITT population).
In conclusion, current evidence suggests that
there is an increase in both the number of oocytes
retrieved and clinical pregnancy rates in poor
responders treated with rec-hLH in addition to
rec-hFSH.

16.4.4 Hyporesponders
The concept of “hypo-response” to COS has been
proposed to identify those at ﬁrst hand good
prognosis normogonadotropic young womenwith normal ovarian reserve who turn out torequire high amounts of rec-hFSH (e.g., >2500 IU
total dose) to obtain an adequate number (i.e.,>4) of oocytes retrieved [64–67]. Such normoovulatory and normogonadotropic women differ
from classical poor responders because they areusually young (<39 years) and ovarian biomarkers (AMH/AFC) are within normal ranges.
Although the pathogenesis of hyporesponsiveness to FSH is unknown, it has been speculated
that hypo-response is a genetically determined
condition (see Chapter 14: Pharmacogenomics
Approach to Controlled Ovarian Stimulation).
More speciﬁcally, ovarian resistance to exogenous FSH has been associated with the presenceof at least two genetic variations, including a
polymorphic allele of the LH beta-subunit gene(v-betaLH), which has been shown to havealtered in vitro and in vivo activities, and FSH
receptor (FSH-R) Ser/680 variant [69, 80–83].
Given a possible association between ovarian
resistance to FSH stimulation (hypo-response)s.esteves@androfert.com.br

16

The Role of LH in Controlled Ovarian Stimulation

and a genetically determined less bioactive LH
molecule or FSH-R dysfunction, several investigators have examined the roles of exogenous LH
activity supplementation and increased FSH
doses on ART cycle outcome.
The role of LH supplementation and increased
FSH doses in hyporesponders were evaluated by
Ferraretti and colleagues, who conducted an RCT
involving 184 patients (age <38 years) undergoing COS for IVF after pituitary desensitization[67]. Hyporesponsiveness to rec-hFSH wasdeﬁned by the observation of a steady follicular
growth (>10 antral follicles ≥8 mm in diameter)
and estradiol levels (≥100 pg/mL) between stimulation days 7–10 despite continuous rec-hFSH
administration. Upon reaching this stage, patientswere randomized to receive (1) an increased rechFSH dose alone (max 450 IU/daily; n = 54), (2)LH activity supplementation with rec-hLH(75 IU/day or 150 IU/day) in addition to anincreased FSH dose (n = 54), and (3) LH activity
supplementation with hMG in addition to anincreased rec-hFSH dose (n = 26). Fifty-four agematched women with normal responses to COS
were included as a control group. The average
number of oocytes retrieved was signiﬁcantly
lower in hyporesponders treated with rec-hFSH
step-up (8.2) versus the other three groups (11.1,
10.9, 9.8), respectively. Pregnancy rates were signiﬁcantly higher in the group treated with rechLH plus increased rec-hFSH dose (54.4 %)compared with both the patients receiving rechFSH alone (24.4 %) and hMG (11 %; p < 0.05).
Pregnancy rates in the group of women receiving
rec-hLH supplementation and an increased rechFSH dose were not different from controls(41 %). Although live birth rates in both rec-hLH(40.7 %) and control (37 %) groups were twofold
higher than the other two groups (22 % and 18 %,respectively), the difference did not reach statistical signiﬁcance.
The role of rec-hLH supplementation per sein hyporesponders was evaluated by De Placido
and colleagues, who conducted a multicenter
RCT involving a total of 117 IVF/ICSI cycles[66]. Hyporesponders (age <37 years, basal FSH≤10 IU/l) were deﬁned by the presence of low
serum estradiol levels (below 180 pg/mL) and at

183

least 6 follicles ranging between 6 and 10 mm,
but no follicles over 10 mm on stimulation day
8 with rec-hFSH. After pituitary desensitization and stimulation with a ﬁxed dose (225 IU/day) of rec-hFSH for the ﬁrst 8 days, the patientswere randomized to receive either an additional 150 IU/day of rec-hLH supplementation(n = 65) or an increase in the daily dose of rechFSH by 150 IU/day (n = 65; rec-hFSH “stepup” protocol). An age/BMI-matched populationof “normal responders” (i.e., tripling E2 levels
between stimulation days 5 and 8 and morethan 4 follicles >10 mm on stimulation day 8)
was selected as a control group (n = 130). The
number of oocytes retrieved was signiﬁcantly
higher in the patients who received rec-LH supplementation (9.0 ± 4.3) compared with thosein whom an increased dose of rec-hFSH wasadministered (6.1 ± 2.6; p < 0.01), and the resultsof both aforementioned groups were lower thanthose obtained in the control group (10.49 ± 3.7;
p < 0.05). Implantation and ongoing pregnancy
rates were similar in “hyporesponders” treatedwith rec-hLH and “normal responders” (14.2 %and 32.5 % vs. 18.1 % and 40.2 %, respectively).
Conversely, both parameters were signiﬁcantly
lower (p < 0.05) in “hyporesponders” treated withstep-up rec-hFSH (10.0 and 22.0 %).
In conclusion, these studies reinforced the ideathat hyporesponders beneﬁt from LH supplementation and that hyporesponsiveness to rec-hFSH
could be related to the presence of less bioactive
LH due to speciﬁc genetic-determined variationsin the beta subunit of the LH molecule.

16.4.5 Deeply Suppressed LH Levels
Profound suppression of LH concentrations as aconsequence of GnRH-agonist downregulation
has been found in 7–48 % of normogonadotropic
women undergoing controlled ovarian stimulation[60, 61, 84, 85]. This wide variation might be justiﬁed by the type and mode of GnRH-agonist
action. Intranasal administration of buserelin
resulted in signiﬁcantly less depressed levels of
mid-follicular LH levels compared with the
subcutaneous route. Moreover, the inhibitorys.esteves@androfert.com.brS.C. Esteves and C. Alviggi

184

effect on ovarian steroidogenesis and folliculardevelopment is more evident with the more potent
buserelin than with leuprolide acetate [61, 85].
Early studies have suggested that ovarian
response and IVF cycle outcome are negatively
impacted when mid-follicular serum LH levels
are below a certain threshold (between 0.5 and
0.7 UI/L) after downregulation with GnRH agonists and ovarian stimulation with FSH monotherapy [59–63, 84]. Westergaard and colleagues,
retrospectively analyzing 200 normogonadotropic
women, reported that as many as 49 % of thosestimulated with rec-hFSH under pituitary suppression with buserelin acetate (0.5 mg SC daily)
for 14 days achieved very low concentrations of
LH (<0.5 IU/L) in the mid-follicular phase, albeit
GnRH-a dose was reduced to 0.2 mg SC per day
during ovarian stimulation [60]. In comparison
with the normal LH group, these women had serum
estradiol concentrations signiﬁcantly lower on Sd8(1349 ± 101 vs. 2908 ± 225 pmol/L; p < 0.001).
Although the proportion of patients with a positive
pregnancy test was similar in the two groups (30 %vs. 34 % per started cycle in the low and normal
LH groups, respectively), a ﬁvefold higher risk ofearly pregnancy loss was observed in the low LH
group (45 % vs. 9 %; p < 0.005). In another study,
Fleming and colleagues found that 26 % of women
treated with highly puriﬁed or recombinant FSH
and GnRH agonist (type and dose not reported)
had suppressed LH concentration (≤0.7 IU/l) on
Sd7 [84]. Patients with suppressed LH had lower
estradiol concentrations (p = 0.001) irrespectiveof whether the FSH is derived from puriﬁed urinary or recombinant sources. However, the negative impact on cycle outcome (longer treatment
duration combined with a reduced oocyte yield)
was observed only in women treated with urinary
FSH, thus indicating that the more potent recombinant FSH treatment could overcome the impactof LH suppression upon gross ovarian response.
Furthermore, no effect upon pregnancy outcome
was observed irrespective of the level of LH suppression and type of FSH preparation. Humaidan
and colleagues also studied the effect of LH levels on stimulation day 8 on ovarian response andpregnancy outcome [61]. The authors retrospectively analyzed 207 normogonadotropic women

receiving pituitary downregulation with buserelin
acetate (0.8 mg SC daily until pituitary downregulation and 0.4 mg/day during ovarian stimulation)and ovarian stimulation with rec-hFSH. LH levels
on Sd8 were directly related to estradiol levels andinversely related to the total consumption of exogenous FSH and duration of gonadotropin stimulation (p < 0.002). In their study, however, only 12 %of the patients showed LH levels <0.5 IU/L. Whilethe number of retrieved oocytes was not affected by
LH suppression, the frequency of fertilized oocytes
was signiﬁcantly lower in the group with profoundLH suppression (p < 0.05). Likewise in the studyof Fleming and cols., pregnancy and implantation
rates were not signiﬁcantly affected by profoundmid-follicular LH suppression. Taken together,
these aforementioned studies indicate that deeply
suppressed LH levels in GnRH agonist-treated
women have a signiﬁcant impact on the ovarian
response during ovarian stimulation, but its impact
on pregnancy outcome is controversial.
Contrary results have been reported by Balaschand colleagues studying 144 infertile women
undergoing IVF/intracytoplasmic sperm injection(ICSI) treatment, in whom pituitary desensitization
was carried out by the administration of leuprolide
acetate (1 mg SC daily, then reduced to 0.5 mg after
downregulation was conﬁrmed) [85]. Using a
receiver-operating characteristic (ROC) analysis,
the authors showed that the serum LH concentration on Sd7 was unable to discriminate between
conception and non-conception cycles (AUC = 0.52;
95 % CI: 0.44–0.61). In this study, only 7 % of thepatients had mid-follicular LH serum concentration <0.5 IL/L, and no signiﬁcant differences were
found with respect to ovarian response, number ofoocytes retrieved, IVF/ICSI outcome, implantation, and the outcome of pregnancy between thesepatients and those with normal LH on Sd7.
In conclusion, conﬂicting evidence exists
regarding the impact of deeply suppressed levels of
mid-follicular serum LH levels on ovarian responseto stimulation with rec-FSH. Current data indicatethat the choice of GnRH-a plays a role in the frequency of patients exhibiting profound mid-follicular LH suppression. Whether these women would
beneﬁt from supplementation with LH activity during ovarian stimulation remains to be proven.

s.esteves@androfert.com.br

16

The Role of LH in Controlled Ovarian Stimulation

16.5

Gonadotropin Preparations
Containing LH Activity

Currently, there are three commercially available
gonadotropin preparations containing LH activity: (1) urinary hMG, in which LH activity isdependent on hCG rather than LH, (2) pure LH
glycoprotein produced by recombinant technology (lutropin alfa), and (3) a combination of FSH(follitropin alfa) and LH (lutropin alfa) in a ﬁxed
ratio of 2:1 also manufactured by recombinant
technology (Sect. 16.1) [3].

16.5.1 Menotropin
Menotropin, or human menopausal gonadotropin(hMG), was ﬁrst extracted from the urine of postmenopausal women in 1949 [2]. Early preparations contained varying amounts of FSH, LH, and
hCG in only 5 % pure forms [1–3]. Improvementsin the puriﬁcation techniques standardized FSH
and LH activities to 75 IU for each type of gonadotropin in 1963, as measured by standard in vivo
bioassays (Steelman–Pohley assay). Human
menopausal gonadotropin preparations have both
FSH and LH activity, but the latter is primarily
derived from the hCG component present in postmenopausal urine and concentrated during puriﬁcation [2, 86, 87, 88]. Sometimes hCG is added toachieve the desired amount of LH-like biologicalactivity [2]. In 1999, puriﬁed hMG gonadotropins
were introduced, allowing its subcutaneous (SC)administration [2, 3]. At present, both conventional hMG and highly puriﬁed hMG (HP-hMG)
are commercially available in vials containing
lyophilized powder of FSH and LH at 1:1 ratio [3].
The enhanced purity of HP-hMG enabled subcutaneous delivery [3].

185

for human LH alpha and beta subunits are incorporated into the nuclear DNA of Chinese hamster
ovary (CHO) cells via a plasmid vector [2, 3, 88].
As a result, a master LH-producing cell bank isbuilt [88, 89]. A working cell bank is then made by
growing cells in culture ﬂasks, which are afterwards combined with a suspension of microcarrierbeads and transferred to a bioreactor vessel with
continuous culture media infusion. The cell culture supernatant medium, containing the proteinssecreted by the cells, is collected from the bioreactor. The harvested “crude LH” is then puriﬁed bychromatography, followed by ultraﬁltration. Eachpuriﬁcation step is rigorously controlled in orderto ensure batch-to-batch consistency of the ﬁnal
puriﬁed product that is the recombinant human LH(rec-hLH) [3].
Lutropin alfa is highly pure and has high biological activity (9000 IU/mg protein) [3, 90]. It ispresented in vials of 82.5 IU lyophilized pure glycoprotein powder to be reconstituted with diluent
before administration using a conventional
syringe and needle (75 IU of lutropin alfa is delivered per vial). Lutropin alfa is intended for subcutaneous daily injections, which represents animportant gain for patients as better tolerability(lower pain at injection site) has been reportedwith SC injections compared with the intramuscular route. Importantly, SC injections allow selfadministration that is more convenient and lesstime consuming as patients need fewer visits tothe clinic or hospital for injections [91, 92]. Dueto the relatively short half-life of LH, daily injections of lutropin alfa are needed during the stimulation period [3, 90]. After each injection, terminal
half-life is reached within 10–12 h, and then LH
levels decline until the next injection.

16.5.2 Lutroprin Alfa

16.5.3 Follitropin Alfa
in Combination
with Lutropin Alfa

Lutroprin alfa was introduced in the market in theyear 2000 intended for promoting ovarian stimulation in women with WHO type I anovulation. The
manufacturing process of lutropin alfa involves
recombinant technology in which the genes coding

A preparation containing both rec-hFSH (follitropin alfa) and rec-hLH (lutropin alfa) at 2:1
ratio was launched in 2007 [93]. The 2:1 ratio of
FSH and LH in a ﬁxed dose combination was
obtained by recombinant technology and vials.esteves@androfert.com.brS.C. Esteves and C. Alviggi

186
Table 16.1 Differences in LH activity among gonadotropin preparations

Lutropin alfa
Follitropin
alfa + lutropin alfa
2:1 ratio
HP-hMG

Purity (LH content)
>99 %
>99 %

FSH activity(IU/vial)
0150

LH activity(IU/vial)75a
75

hCG content(IU/vial)––

Speciﬁc activity(LH/mg protein)
9000
9000

Unknownb

75

75b

~8

–

1 μg of lutropin alfa = 22 IU
Derives primarily from the hCG component, which preferentially is concentrated during the puriﬁcation process andsometimes was added to achieve the desired amount of LH-like biological activity
HP-hMG highly puriﬁed human menopausal gonadotropinabﬁlling using protein mass (FbM). The use of FbM
as opposed of ﬁlled-by-bioassay was possible
due to the speciﬁc activity; isoform distribution
and sialylation proﬁle of both gonadotropins arehighly consistent among manufactured batches[88]. It is intended for subcutaneous daily injections and is presented in vials of lyophilized pureglycoprotein powder to be reconstituted withdiluent before administration using a conventional syringe and needle (150 IU of follitropin
alfa and 75 IU of lutropin alfa is delivered pervial). The results of two phase I, randomized,
crossover studies demonstrated bioequivalence
between rec-hFSH and rec-hLH administeredalone or in ﬁxed 2:1 combination, thus allowing
administration of both recombinant gonadotropins in a single injection [94].

16.6

Differences in LH Activity
Between rec-hLH and hMG
Preparations

Recombinant LH has three major differencescompared with hMG preparations. First, rechLH has a better quality and safety proﬁle compared with hMG [3, 90]. High purity and speciﬁc
activity are common features of gonadotropin
preparations manufactured using recombinant
technology. Each product batch of recombinant
LH is routinely characterized and controlledusing physicochemical techniques, including
size-exclusion high-performance liquid chromatography (SE-HPLC), which allows assessment of both the integrity and the amount ofglycoproteins, and isoelectric focusing (IEF)
and glycan mapping, which characterize protein glycoforms present in each preparation [95,96]. Conversely, the manufacturing process ofurine-derived gonadotropins is less stringent, as
urine is pooled and the donor source cannot be
fully traced. As the pool is constantly changing,standardization is difﬁcult to ascertain [97–99].
Although sophisticated puriﬁcation techniques
are currently available, which allow the safe
clinical use of urinary formulations, extraneous
urinary proteins may account for more than 30 %of the protein content in high-puriﬁed hMG products (Table 16.1) [3, 100].
Second, rec-hLH is associated with better dose
precision due to ﬁll-by-mass (FbM) technologythat virtually eliminates batch-to-batch variation[2, 3, 100, 101]. The conventional method used toquantify the glycoprotein activity in gonadotropin
products is the Steelman–Pohley assay, which isan in vivo rat bioassay. As well as being costly andsubject to ethical concerns related to the use of animals, this technique has an inherent variability of
up to 20 % [101, 102]. In 2003, Driebergen andBaer demonstrated the batch-to-batch consistencyof follitropin alfa in terms of speciﬁc activity, isoform pattern, and sialylation proﬁle [101]. The
authors showed that there was a constant relationship between FSH mass and its biological activity.
Following these observations, a new method wasdeveloped to calibrate each batch of follitropin
alfa, and also lutropin alfa, using SE-HPLC, whichmeasures glycoprotein content by protein mass.
This technique has enabled lutropin alfa to be
ﬁlled and released on the basis of mass (75 IU ofs.esteves@androfert.com.br

16

The Role of LH in Controlled Ovarian Stimulation

187

Table 16.2 Structural characteristics, half-life in serum, and downstream effects of LH and hCG following receptor
bindingAmino acid number
Alpha subunit
Beta subunit
N-linked glycosylation sites
Alpha subunit
Beta subunit
O-linked glycosylation sites
Carboxyl terminal segment
Half-life (hours)
Initial, range of mean
Terminal, range of mean
Response
ED50 (pM)aTime to maximal cAMP accumulationa
ERK 1/2 activationb
AKT activationb
CYP19A1 expression in presence of ERK1/2 pathway blockadeb

LH

hCG

92
121

92
145

2
1
–
Nonexistent2
2
4
Present

0.6–1.3
9–12

3.9–5.5
23–31

530.0 ± 51.2
10 minStrongStrongIncreased

107.1 ± 14.3
1h
Weak
MinimalUnaffected

Initial half-life (distribution): time for the plasma concentration to decrease steeply because of the distribution into
tissues
Terminal (elimination) half-life = time that it takes for the concentration in blood plasma of a substance to reach one-halfof its steady-state value
ERK extracellular signal-regulated kinases, AKT protein kinase B, CYP19A10 cytochrome P450, family 19, subfamily A1aMedian effective dose to produce a response 50 % of the COS-7/LHCGR cells, which constitutively express LH
receptorsb
Effect on human granulosa cells

LH assessed by the Steelman–Pohley assay corresponds to 3.4 μg of lutropin alfa), with dose variability of only 2 % [90, 101].
Third, LH activity is derived from pure LH
glycoprotein unlike hMG, in which hCG is concentrated during puriﬁcation or added to achieve
the desired amount of LH-like biological activity[2, 3, 90]. LH and hCG differ in the compositionof their carbohydrate moieties which, in turn,affect bioactivity and half-life. Although hCGamino acid sequence is similar to that of LH, anotable difference is the presence of a long carboxyl terminal segment with 24 AA containing
four sites of O-linked oligosaccharides [3, 103](see Fig. 16.1). Furthermore, hCG beta subunits
contain two sites of N-linked glycosylation compared with a single site in LH. Due to the higher
number of both glycosylation sites and sialic acidresidues (approximately 20) than LH, hCG
exhibit a markedly longer terminal half-life.
After administration, recombinant human LH iseliminated with a terminal half-life of 10–12 h incontrast to 23–31 h of hCG [10, 104] (Table 16.2).

16.6.1 Differences Between LH
and hCG: Evidence
from In Vitro Studies
Although both human LH and hCG act on the same
LH/hCG receptor (LH-R), evidence from in vitro
models indicates that LH receptors differentiate
between LH and hCG coupling. While LH exclusively stimulate the targeted LH-R by cis-activation, hCG is also capable of inducing transactivation,
thus affecting the kinetics of cAMP production anddownstream ERK1/2- and AKT-pathway activation [105, 106]. Using equimolar concentrations of
LH and hCG in human granulosa cells obtained
from women undergoing oocyte retrieval for ART,
Casarini and colleagues have shown that hCG is
ﬁvefold more potent in vitro than LH at the receptor

s.esteves@androfert.com.brS.C. Esteves and C. Alviggi

188

level based on the measurement of intracellularcAMP [105]. Using equipotent doses of LH andhCG, however, the aforementioned authors showedthat accumulation of intracellular cAMP by LH
was signiﬁcantly faster, with maximal activationachieved in 10 minutes, while by hCG the samelevels of the maximal stimulation were attained
only after 60 min of stimulation (see Table 16.2).
Interestingly, LH and hCG were equipotent interms of progesterone production in spite of overalllower cAMP levels after LH stimulation. Despite
being mainly dependent on the cAMP/PKApathway [107], progesterone production in preovulatory GCs may involve other signaling pathways
modulated by ERK1/2 and AKT [108], including[109] molecules of the EGF family such as neuregulin 1 and amphiregulin [34, 110].
In additional experiments using the samein vitro model, Casarini and cols. also evaluated the effects of LH and hCG at activating the
ERK1/2 and AKT pathways. ERK and AKT arecell cycle regulators; while AKT is involved incell signaling leading to cell survival (by blocking apoptosis), ERK represents a range of cell
proteins that communicate a signal from a receptor on the cell surface to the DNA in the nucleus.
Stimulation with equimolar concentrations of
LH resulted in a strong, rapid (10 min), and sustained (45 min) activation of ERK1/2, while hCG
induced a much weaker and short-lived stimulation, reaching signiﬁcance only at 10 min. As far
as AKT is concerned, LH provoked a substantial
increase in AKT between 10 and 30 min, while
hCG stimulation at same doses was virtuallynonexistent. Given the different intracellular signaling of hLH and hCG on acute ERK and AKT
activation, these authors assessed whether the
expression of genes known to be under LH and/or
hCG control, epiregulin (AREG) and neuregulin
1 (NRG1) and of CYP19A1 (aromatase), would
be differentially affected by the type of gonadotropin. While both LH or hCG stimulation resulted
in a marked stimulation of the expression of suchgenes, LH was signiﬁcantly more potent than hCG
on AREG. Epiregulin may play a role in the ovulatory process and oocyte maturation [108, 111]
exerted via both ERK- and AKT-pathway activation [34, 112]. In conclusion, LH and hCG action

on the same receptor results in quantitatively
and qualitatively different intracellular signaling. While equimolar concentrations of LH andhCG possess different in vitro potency (in termsof cAMP), equipotent concentrations of LH andhCG stimulate intracellular cAMP accumulation
with signiﬁcantly different kinetics. Moreover, LH
is more potent than hCG on the ERK and AKT
pathways and elicits different kinetic response.
The investigation of the functional role of thecAMP, ERK, and AKT signaling pathways inhuman fertility has revealed that LH/hCG stimulation of the same receptor results in activation ofdifferent, complex signal transduction pathways
and molecules [111–113]. In vitro activation ofcAMP-pathway by gonadotropins is traditionallyassociated with structural changes, consisting incell-rounding [114, 115], apoptotic events [115–117] and in the prevention of meiosis resumptionof the oocyte [118]. In contrast, gonadotropindependent activation of antiapoptotic pathways[34, 119] and proliferative effects [120] seems to
be mediated by ERK1/2 and AKT, and reductionof ERK1/2 signaling activates apoptotic signals
in the GCs [121]. Taken together, these results
indicate that hCG and hLH action on the regulation of cell cycle and apoptosis in granulosa cellmight be divergent and/or dependent on which
signal transduction pathway is activated. This isespecially relevant in inﬂuencing the cell fate
during folliculogenesis, when the activation ofdifferent signal transduction pathways mediates adelicate balance between pro- and antiapoptotic
signals [122]. While the in vivo effects of the differential activation of the various pathways
remain to be investigated, the nonequivalence of
LH and hCG deserves consideration in the application of therapeutic strategies involving LH
activity supplementation in COS protocols.

16.6.2 Differences Between LH
and hCG: Evidence
from Clinical Studies
It has been shown that the expression of the LH
receptor gene, as well as genes involved in thebiosynthesis of cholesterol and steroids in granulosa

s.esteves@androfert.com.br

16

The Role of LH in Controlled Ovarian Stimulation

cells (CYP11A activity decreased by 2.4-fold), islower in patients treated with hMG preparations
compared with those treated with FSH preparations [123]. Such effects are caused by a constantligand exposure during the follicular phase due tolong half-life and high receptor binding afﬁnity
of hCG. In animal models, downregulation of LH
receptors is maintained for up to 48 h after hMGadministration [124]. These ﬁndings indicate that
the GCs have lower LH-induced cholesterol
uptake, a reduction in the de novo cholesterolsynthesis, and a reduction in steroid synthesis
and thus could explain the observed lower serum
progesterone levels achieved in patients treatedwith hMG compared with FSH [123, 125].
In a study prospectively evaluating 60 normogonadotropic women who, when undergoing
induction of multiple follicular growth, showed
an insufﬁcient ovarian response in terms of follicular growth (deﬁned as “low responders”),
Ruvolo and colleagues examined the impact of
LH supplementation on cumulus cell apoptosis[126]. On stimulation day 8, one group wastreated with rec-hFSH combined with rec-hLH,
while the other was stimulated with rec-hFSH
alone. Terminal deoxynucleotidyl transferasemediated digoxigenin-deoxyuridine-triphosphate(dUTP) nick-end labeling (TUNEL) assay andanti-caspase-3 cleaved immunoassay were used
to measure apoptosis in the cumulus cells. A statistically signiﬁcant increase in the number of
immature oocytes was observed in the grouptreated with rec-hFSH alone (2.33 in the rechFSH group vs. 0.58 in the rec-hLH group;p < 0.01). In contrast, apoptosis markers werelower in the group who received LH supplementation by rec-hLH. A lower rate of cells withchromatin fragmentation (TdT, 18.2 % vs.
12.1 %) and lower presence of caspase-3 cleaved(17.0 % vs. 11.0 %) were observed in the rechLH group compared with the rec-hFSH group.
Implantation rates were signiﬁcantly higher inthe rec-hLH group (15.6 %) compared with the
rec-hFSH group (12.5 %, p < 0.01). The authors
concluded that supplementation with rec-hLH
reduced the number of immature oocytes collected after pickup. Furthermore, they speculated
that the increase in implantation rate might be189correlated with the reduction of apoptosis seen in
the cumulus cells of patients treated with rechLH, due to a direct action of rec-hLH on the
cumulus and granulosa cells, or because of theparacrine effect mediated by secreting factors in
the theca and oocyte cells. Thus, maintaining
their physiological function for a longer time,
cumulus cells are better able to support nuclear
and cytoplasmic maturation of the oocyte until
ovulation, thus allowing the collection of oocyteswith better “intrinsic” qualities that are necessary
for sustaining fertilization and the early phases ofembryogenesis. Hence, if cumulus cells are preserved from apoptotic processes, the oocyte
receives no molecular signal able to activate
apoptotic pathways [126].
The clinical implications of the aforementioned observations have been investigated by“tail to tail” comparison between hMG and recLH preparations. An open-label RCT in 2012
compared HP-hMG and a ﬁxed combination of
rec-FSH and rec-hLH in 35 women with hypogonadotropic hypogonadism. Eighteen patients
received 150 IU hMG-HP (150 IU FSH + 150 IU
LH-like activity) and seventeen received 150 IU
rec-hFSH/75 IU rec-hLH daily for a maximum of
16 days. The proportion of patients reaching ovulation did not differ between the groups (70 % vs.
88 %, respectively), but the pregnancy rate wassigniﬁcantly higher in those treated with the combination of recombinant gonadotropins (55.6 %vs. 23.3 %; p = 0.01) [127]. In another RCT,Pacchiarotti and colleagues enrolled 122 womenwith low baseline endogenous LH levels(<1.2 IU/L) in the presence of normal FSH levels
undergoing IVF. The patients were treated with adownregulation protocol consisting of triptorelin
0.1 mg at day 21 of the cycle and were randomized to receive an ovarian stimulation with 225 IU/
day of either HP-hMG or rec-FSH plus rec-hLH
in a 2:1 ratio. Fewer days of stimulation (10.9 ± 1.1
vs. 14.1 ± 1.6; p = 0.013) and a higher number of
retrieved oocytes (7.8 ± 1.1 vs. 4.1 ± 12; p = 0.002)
were noted in the group that received follitropin
alfa + lutropin alfa 2:1 compared with the group
who received HMG. However, differences were
not observed in estradiol levels on hCG day(1987 ± 699 pg/mL vs. 2056 ± 560 pg/mL),s.esteves@androfert.com.brS.C. Esteves and C. Alviggi

190

pregnancy rates per cycle (28.3 % vs. 29.3 %),and implantation rates (12.1 % vs. 12.2 %),despite higher cancelation rates due to excessive
response in women receiving follitropin + lutropin
alfa (11.1 % vs. 1.7 %; p = 0.042), which therefore
indicates that the latter is a more potent preparation for COS [128].
Furthermore, the German experience withthe use of rec-hLH compared with hMG in
daily ART practice was recently reported byanalyzing data from the National IVF Registry(DIR), in which patients undergoing IVF fromapproximately 85 % of the German IVF centers
are prospectively enrolled [129]. A total of 4719
women, 1573 per group, matched by age, bodymass index, indication, and number of previous
ART cycles, treated with either rec-hFSH andrec-hLH in a ﬁxed 2:1 ratio or hMG, either alone
or in combination with rec-hFSH, after downregulation in a long GnRH-agonist protocol, wasanalyzed. The mean gonadotropin consumption(in ampoules of 75 IU) was signiﬁcantly lower inthe group treated with the ﬁxed combination of
rec-hFSH and rec-hLH (34.3) compared with thetwo hMG groups (hMG alone: 36.4, p < 0.001;
hMG in combination with rec-hFSH: 46.3,
p < 0.001). Pregnancy rates per cycle (25.5 % vs.
21.5 %, p = 0.006; 25.5 % vs. 21.7 %, p = 0.02) andper embryo transfer (31.3 % vs. 26.0 %, p = 0.02;
31.3 % vs. 25.6 %, p = 0.008) and implantation
rate per embryo transferred (19.0 % vs. 14 %in both pairwise comparisons, p < 0.001) werehigher in the group treated with the ﬁxed combination of rec-hFSH and rec-hLH comparedwith the aforesaid hMG groups, respectively.
Lastly, in 2013, a crossover study evaluated 33
patients using HP-hMG in their ﬁrst IVF cycle
and 2:1 rec-hFSH plus rec-hLH in their second
IVF attempt [130]. Estradiol levels on the dayof hCG (2633 ± 871 vs. 2101 ± 816; p < 0.05)
and the number of oocytes retrieved (9.8 ± 3.3
vs. 7.3 ± 3.1; p < 0.01) were higher in the groupthat received the 2:1 rec-hFSH plus rec-hLH
formulation. Despite implantation and clinicalpregnancy rates per started cycle were not different between the groups (29.6 % and 48.4 %,respectively, for hMG and 28.4 % and 48.4 %for rec-hFSH plus rec-hLH), 2/3 of the patientsin rec-FSH + rec-LH group (vs. 1/3 hMG group)
would have frozen embryos to transfer if a freshtransfer failed.
Despite being developed for stimulation offollicular growth in women with severe LH andFSH deﬁciency, the 2:1 formulation of follitropin alfa and lutropin alfa has expanded to normogonadotropic women undergoing ART. In a
3-year, multicenter, open-label, observational,
post-marketing surveillance study involving
2200 German women (21–45 years) undergoing
ART, the most common reasons for physicians
to prescribe the 2:1 formulation of follitropin
alfa and lutropin alfa were poor ovarian response(39.4 %), low baseline LH level (17.8 %), age(13.8 %), and low baseline E2 level (7.3 %) [131].
Recently, a cost-effectiveness model compared
rec-FSH + rec-hLH and HP-hMG for ovulationinduction in hypogonadotropic hypogonadal
women, according to the Italian Health Service
perspective, in which only direct costs (drugs,
specialist visits, patient examinations, and hospitalizations) are included [132]. A Markov
model was developed, considering the probability of pregnancy and miscarriage in three cyclesof therapy. In that model, the patients started thetherapy with recombinant or urinary gonadotropins following pregnancy evaluation. If a woman
became pregnant, the possibility of miscarriage
was considered. Women who did not become
pregnant during the ﬁrst series of treatment or
had a miscarriage underwent a second cycle oftherapy, maintaining the same treatment of theprevious cycle. The same process was applied tothe third cycle. Consumption of gonadotropins
and outcome of HP-hMG and rec-hFSH + rechLH cycles were based on the study by Carone
and cols [127]. Rec-hFSH + rec-hLH was associated with a higher acquisition cost (€3453.50)
and higher efﬁcacy (0.87) compared with
HP-hMG (€2719.70 and 0.50). The average cost
per pregnancy was estimated to be €3990.00 forrecombinant strategy and €5439.80 for urinary
strategy, thus indicating that the combination
therapy with rec-hFSH + rec-hLH is associatedwith a better cost-effectiveness compared to
HP-hMG in the treatment of infertility in hypogonadotropic hypogonadal women.

s.esteves@androfert.com.br

16

The Role of LH in Controlled Ovarian Stimulation

In conclusion, evidence from experimental
and clinical studies indicates that LH activity
driven by hCG and rec-hLH is not equivalent neither at the molecular level nor at the functional
level. COS protocols involving LH supplementation with rec-hFSH appear to be more effective
and efﬁcacious than those with hMG (hCG activity). Nevertheless, large RCTs are needed to conﬁrm these observations.
Conclusions• Ovarian steroidogenesis is the result ofcombined LH and FSH stimulation of thetwo cell types, theca and granulosa, inﬂuenced by autocrine and paracrine factors.
• LH has a pivotal role in follicular development and maturation. LH is crucial in sustaining FSH activity in the granulosa during
intermediate–late stages of folliculogenesis
and is critical for maintaining corpus luteum
function during the luteal phase.
• Exogenous LH activity supplementation is
mandatory in stimulation protocols applied to
women with hypogonadotropic hypogonadism.
• Review of the studies exploring the supplementation of rec-hLH to COS regimens
indicates that, at present, the addition of
rec-hLH to the general population of infertile women undergoing IVF/ICSI cycles
remains controversial.
• Fair evidence indicates that rec-hLH supplementation has a positive effect on cycle outcome of older women (>35 years old),particularly when used from the start of COS.
• Fair evidence indicates that both the number of oocytes retrieved as well as the clinical pregnancy rates are increased in poor
responders undergoing COS with a combination of rec-hLH and rec-hFSH.
• Hyporesponsiveness to rec-hFSH could be
related to the presence of less bioactive LH
due to speciﬁc genetic-determined variationsin the beta subunit of the LH molecule.
Limited data indicate that hyporesponders
beneﬁt from LH supplementation during
COS.
• Conﬂicting evidence exists regarding theimpact of deeply suppressed levels of

191

mid-follicular serum LH levels on ovarian
response to stimulation with recFSH. Current data indicates that the choiceof GnRH-a plays a role on the frequency ofpatients exhibiting profound mid-follicular
LH suppression. Whether these women
would beneﬁt from supplementation with
LH activity during ovarian stimulation
remains to be proven.
• Evidence from experimental and clinicalstudies indicate that LH activity driven by
hCG and rec-hLH is not equivalent neither
at the molecular level nor at the functional
level. COS protocols involving LH supplementation with rec-hLH, particularly using
a ﬁxed 2:1 combination of rec-hFSH andrec-hLH, appear to be more effective andefﬁcacious than those with hMG (hCG
activity). Nevertheless, large RCTs areneeded to conﬁrm these observations.

References
1. Beall SA, DeCherney A. History and challenges surrounding ovarian stimulation in the treatment ofinfertility. Fertil Steril. 2012;97(4):785–801.
2. Lunenfeld B. Historical perspectives in gonadotrophin therapy. Hum Reprod Update. 2004;10(6):453–67.
3. Leão RB, Esteves SC. Gonadotropin therapy inassisted reproduction: an evolutionary perspectivefrom biologics to biotech. Clinics (Sao Paulo).
2014;69(4):279–93.
4. Alviggi C, Mollo A, Clarizia R, De Placido
G. Exploiting LH in ovarian stimulation. Reprod
Biomed Online. 2006;12(2):221–33.
5. Speroff L, Fritz MA. Chapter 5: Neuroendocrinology.
In: Speroff L, Fritz MA, editors. Clinical gynecologic endocrinology and infertility. Philadelphia:
Lippincott Williams & Wilkins; 2005. p. 295–6.
6. Vaitukaitis JL, Ross GT, Braunstein GD, Rayford
PL. Gonadotropins and their subunits: basic andclinical studies. Recent Prog Horm Res. 1976;32:
289–331.
7. Ulloa-Aguirre A, Espinoza R, Damian-Matsumura
P, Chappel SC. Immunological and biological potencies of the different molecular species of gonadotrophins. Hum Reprod. 1988;3(4):491–501.
8. Wide L, Naessén T, Sundström-Poromaa I, Eriksson
K. Sulfonation and sialylation of gonadotropins inwomen during the menstrual cycle, after menopause,and with polycystic ovarian syndrome and in men.
J Clin Endocrinol Metab. 2007;92(11):4410–7.

s.esteves@androfert.com.brS.C. Esteves and C. Alviggi

192
9. Ulloa-Aguirre A, Midgley Jr AR, Beitins IZ,
Padmanabhan V. Follicle-stimulating isohormones:
characterization and physiological relevance. Endocr
Rev. 1995;16(6):765–87.
10. Campbell RK. Molecular pharmacology of gonadotropins. Endocrine. 2005;26(3):291–6.
11. Combarnous Y. Molecular basis of the speciﬁcity ofbinding of glycoprotein hormones to their receptors.
Endocr Rev. 1992;13(4):670–91.
12. Costagliola S, Urizar E, Mendive F, Vassart G.
Speciﬁcity and promiscuity of gonadotropin receptors. Reproduction. 2005;130(3):275–81.
13. Hsueh AJ, Adashi EY, Jones PB, Welsh Jr TH.
Hormonal regulation of the differentiation of cultured ovarian granulosa cells. Endocr Rev.
1984;5(1):76–127.
14. Jeppesen JV, Kristensen SG, Nielsen ME, et al.
LH-receptor gene expression in human granulosa andcumulus cells from antral and preovulatory follicles.
J Clin Endocrinol Metab. 2012;97(8):E1524–31.
15. Speroff L, Fritz MA. Chapter 2: Hormone biosynthesis, metabolism, and mechanisms of action.
Steroidogenesis. In: Speroff L, Fritz MA, editors.
Clinical gynecologic endocrinology and infertility.
Philadelphia: Lippincott Williams & Wilkins; 2005.
p. 109–16.
16. Young JM, McNeilly AS. Theca: the forgotten cellof the ovarian follicle. Reproduction. 2010;140(4):489–504.
17. Falck B. Site of production of oestrogen in rat ovary
as studied in microtransplants. Acta Physiol Scand
Suppl. 1959;47(163):1–101.
18. Miller WL. Steroidogenic enzymes. Endocr Dev.
2008;13:1–18.
19. Wickenheisser JK, Nelson-DeGrave VL, McAllister
JM. Human ovarian theca cells in culture. Trends
Endocrinol Metab. 2006;17(2):65–71.
20. Smyth CD, Miró F, Whitelaw PF, Howles CM,
Hillier SG. Ovarian thecal/interstitial androgen synthesis is enhanced by a follicle-stimulating hormonestimulated paracrine mechanism. Endocrinology.
1993;133(4):1532–8.
21. Whitelaw PF, Smyth CD, Howles CM, Hillier
SG. Cell-speciﬁc expression of aromatase and LH
receptor mRNAs in rat ovary. J Mol Endocrinol.
1992;9(3):309–12.
22. Speroff L, Fritz MA. Chapter 6: Regulation of themenstrual cycle. In: Speroff L, Fritz MA, editors.
Clinical gynecologic endocrinology and infertility.
Philadelphia: Lippincott Williams & Wilkins; 2005.
p. 348–83.
23. Coutts JRT, Gaukroger JM, Kader AS, et al.
Steroidogenesis by the human Graaﬁan follicle. In:
Coutts JRT, editor. Functional morphology of thehuman ovary. Lancaster: MTP; 1981. p. 56–72.
24. Filicori M, Cognigni GE, Taraborrelli S, et al.
Luteinizing hormone activity supplementation
enhances follicle-stimulating hormone efﬁcacy andimproves ovulation induction outcome. Clin
Endocrinol Metab. 1999;84(8):2659–63.

25. Couzinet B, Brailly S, Bouchard P, Schaison
G. Progesterone stimulates luteinizing hormone
secretion by acting directly on the pituitary. J Clin
Endocrinol Metab. 1992;74(2):374–8.
26. Zeleznik AJ, Hillier SG. The role of gonadotropinsin the selection of the preovulatory follicle. Clin
Obstet Gynecol. 1984;27(4):927–40.
27. Goff AK, Armstrong DT. Stimulatory action of
gonadotropins and prostaglandins on adenosine-3′,5′monophosphate production by isolated rat granulosa
cells. Endocrinology. 1977;101(5):1461–7.
28. Campbell BK, Dobson H, Baird DT, Scaramuzzi
RJ. Examination of the relative role of FSH and LH
in the mechanism of ovulatory follicle selection in
sheep. J Reprod Fertil. 1999;117(2):355–67.
29. Sullivan MW, Stewart-Akers A, Krasnow JS, Berga
SL, Zeleznik AJ. Ovarian responses in women to
recombinant follicle-stimulating hormone andluteinizing hormone (LH): a role for LH in the ﬁnal
stages of follicular maturation. J Clin Endocrinol
Metab. 1999;84(1):228–32.
30. Vegetti W, Alagna F. FSH and folliculogenesis: fromphysiology to ovarian stimulation. Reprod Biomed
Online. 2006;12(6):684–94.
31. Huang ZH, Clayton PE, Brady G, Morris ID. Insulinlike growth factor-I gene expression in humangranulosa-lutein cells. J Mol Endocrinol. 1994;12(3):283–91.
32. Zhou J, Bondy C. Anatomy of the human ovarian
insulin-like growth factor system. Biol Reprod.
1993;48(3):467–82.
33. Peluso JJ, Pappalardo A, Fernandez G. Basic ﬁbroblast growth factor maintains calcium homeostasis
and granulosa cell viability by stimulating calcium
efﬂux via a PKC delta-dependent pathway.
Endocrinology. 2001;142(10):4203–11.
34. Ben-Ami I, Armon L, Freimann S, Strassburger D,
Ron-El R, et al. EGF- like growth factors as LH
mediators in the human corpus luteum. Hum Reprod.
2009;24(1):176–84.
35. Park J-Y, Su Y-Q, Ariga M, Law E, Jin S-LC, et al.
EGF-like growth factors as mediators of LH actionin the ovulatory follicle. Science. 2004;303(5658):
682–4.
36. Donadeu FX, Ascoli M. The differential effects of the
gonadotropin receptors on aromatase expression inprimary cultures of immature rat granulosa cells arehighly dependent on the density of receptors
expressed and the activation of the inositol phosphatecascade. Endocrinology. 2005;146(9):3907–16.
37. Lindeberg M, Carlström K, Ritvos O, Hovatta
O. Gonadotrophin stimulation of non-luteinizedgranulosa cells increases steroid production and theexpression of enzymes involved in estrogen andprogesterone synthesis. Hum Reprod. 2007;22(2):
401–6.
38. Casper RF, Yen SS. Induction of luteolysis in thehuman with a long-acting analog of luteinizing
hormone-releasing factor. Science. 1979;205(4404):
408–10.

s.esteves@androfert.com.br

16

The Role of LH in Controlled Ovarian Stimulation

39. Sugino N, Kaskida S, Takiguchi S, Karube A, Kato
H. Expression of vascular endothelial growth factor
and its receptors in the human corpus luteum during
the menstrual cycle and in early pregnancy. J Clin
Endocrinol. 2000;85(10):3919–24.
40. Wang TH, Horng SG, Chang CL, Wu HM, Tsai YJ,
Wang HS, Soong YK. Human chorionic gonadotropininduced hyperstimulation syndrome is associatedwith up-regulation of vascular endothelial growth factor. J Clin Endocrinol Metab. 2002;87(7):3300–8.
41. Robinson RS, Nicklin LT, Hammond AJ, Schams D,
Hunter MG, Mann GE. Fibroblast growth factor 2 ismore dynamic than vascular endothelial growth factor A during the follicle-luteal transition in the cow.
Biol Reprod. 2007;77(1):28–36.
42. Stirling D, Waterman MR, Simpson ER. Expressionof mRNA encoding basic ﬁbroblast growth factor(bFGF) in bovine corpora lutea and cultured luteal
cells. J Reprod Fertil. 1991;91(1):1–8.
43. Fraser HM, Dickson SE, Lunn SF, Wulff C, Morris
KD, Caroll VA, Bicknell R. Suppression of luteal
angiogenesis in the primate after neutralization ofvascular endothelial growth factor. Endocrinology.
2000;141(3):995–1000.
44. Berisha B, Stefﬂ M, Amselgruber W, Schams
D. Changes in ﬁbroblast growth factor 2 and its
receptors in bovine follicles before and after GnRH
application and after ovulation. Reproduction.
2006;131(2):319–29.
45. Rao CV. Multiple novel roles of luteinizing hormone. Fertil Steril. 2001;76(6):1097–100.
46. Tesarik J, Hazout A, Mendoza C. Luteinizing hormone
affects uterine receptivity independently of ovarianfunction. Reprod Biomed Online. 2003;7(1):59–64.
47. Licht P, Russu V, Wildt L. On the role of human chorionic gonadotropin (hCG) in the embryoendometrial microenvironment: implications fordifferentiation and implantation. Semin Reprod
Med. 2001;19(1):37–47.
48. Shoham Z, Smith H, Yeko T, O’Brien F, Hemsey G,
O’Dea L. Recombinant LH (lutropin alfa) for thetreatment of hypogonadotrophic women with profound LH deﬁciency: a randomized, double-blind,placebo-controlled, proof-of-efﬁcacy study. Clin
Endocrinol (Oxf). 2008;69(3):471–8.
49. European Recombinant Human LH Study Group.
Recombinant human luteinizing hormone (LH) to support recombinant human follicle-stimulating hormone(FSH)-induced follicular development in LH- and
FSH-deﬁcient anovulatory women: a dose-ﬁnding
study. J Clin Endocrinol Metab. 1998;83(5):1507–14.
50. Loumaye E, Engrand P, Howles CM, O’Dea
L. Assessment of the role of serum luteinizing hormone and estradiol response to follicle-stimulating
hormone on in vitro fertilization treatment outcome.
Fertil Steril. 1997;67(5):889–99.
51. Sills ES, Levy DP, Moomjy M, et al. A prospective,
randomized comparison of ovulation induction
using highly puriﬁed follicle- stimulating hormone
alone and with recombinant human luteinizing

193
hormone in in-vitro fertilization. Hum Reprod.
1999;14:2230–5.
52. Esteves SC, Schertz JC, Verza Jr S, Schneider DT,
Zabaglia SF. A comparison of menotropin, highlypuriﬁed menotropin and follitropin alfa in cycles of
intracytoplasmic sperm injection. Reprod Biol
Endocrinol. 2009;7:111.
53. Kolibianakis EM, Kalogeropoulou L, Griesinger G,
et al. Among patients treated with FSH and GnRH
analogues for in vitro fertilization, is the addition ofrecombinant LH associated with the probability oflive birth? A systematic review and meta-analysis.
Hum Reprod Update. 2007;13(5):445–52.
54. Mochtar MH, Van der Veen, Ziech M, van Wely
M. Recombinant Luteinizing Hormone (rLH) for
controlled ovarian hyperstimulation in assisted
reproductive cycles. Cochrane Database Syst Rev
2007;(2):CD005070.
55. Lehert P, Kolibianakis EM, Venetis CA, Schertz J,
Saunders H, Arriagada P, et al. Recombinant humanfollicle-stimulating hormone (r-hFSH) plus recombinant luteinizing hormone versus r-hFSH alone forovarian stimulation during assisted reproductive
technology: systematic review and meta-analysis.
Reprod Biol Endocrinol. 2014;12:17.
56. Marrs R, Meldrum D, Muasher S, Schoolcraft W,
Werlin L, Kelly E. Randomized trial to compare theeffect of recombinant human FSH (follitropin alfa)
with or without recombinant human LH in women
undergoing assisted reproduction treatment. Reprod
Biomed Online. 2004;8(2):175–82.
57. Hill MJ, Levens ED, Levy G, et al. The use ofrecombinant luteinizing hormone in patients undergoing assisted reproductive techniques withadvanced reproductive age: a systematic review andmeta-analysis. Fertil Steril. 2012;97(5):1108–14.
58. Bosdou JK, Venetis CA, Kolibianakis EM, et al. The
use of androgens or androgen-modulating agents inpoor responders undergoing in vitro fertilization: asystematic review and meta-analysis. Hum Reprod
Update. 2012;18(2):127–45.
59. Esposito MA, Barnhart KT, Coutifaris C, Patrizio
P. Role of periovulatory luteinizing hormone concentrations during assisted reproductive technology cycles
stimulated exclusively with recombinant folliclestimulating hormone. Fertil Steril. 2001;75(3):519–24.
60. Westergaard LG, Laursen SB, Andersen
CY. Increased risk of early pregnancy loss by profound suppression of luteinizing hormone during
ovarian stimulation in normogonadotrophic women
undergoing assisted reproduction. Hum Reprod.
2000;15(5):1003–8.
61. Humaidan P, Bungum L, Bungum M, Andersen
CY. Ovarian response and pregnancy outcome
related to mid-follicular LH levels in women undergoing assisted reproduction with GnRH agonist
down-regulation and recombinant FSH stimulation.
Hum Reprod. 2002;17(8):2016–21.
62. Laml T, Obruca A, Fischl F, Huber JC. Recombinantluteinizing hormone in ovarian hyperstimulations.esteves@androfert.com.brS.C. Esteves and C. Alviggi

194
after stimulation failure in normogonadotrophic
women. Gynecol Endocrinol. 1999;13(2):98–103.
63. Nakagawa K, Ohgi S, Nakashima A, Horikawa T,
Sugiyama R, Saito H. The ratio of late-follicular to
mid-follicular phase LH concentrations efﬁciently
predicts ART outcomes in women undergoing ART
treatment with GnRH-agonist long protocol andstimulation with recombinant FSH. J Assist Reprod
Genet. 2008;25(8):359–64.
64. De Placido G, Mollo A, Alviggi C, et al. Rescue of
IVF cycles by HMG in pituitary down-regulated
normogonadotrophic young women characterized
by a poor initial response to recombinant FSH. Hum
Reprod. 2001;16(9):1875–9.
65. De Placido G, Alviggi C, Mollo A, et al. Effects ofrecombinant LH (rLH) supplementation during controlled ovarian hyperstimulation (COH) in normogonadotrophic women with an initial inadequate responseto recombinant FSH (rFSH) after pituitary downregulation. Clin Endocrinol (Oxf). 2004;60(5):637–43.
66. De Placido G, Alviggi C, Perino A, et al. Recombinanthuman LH supplementation versus recombinanthuman FSH (rFSH) step-up protocol during controlled ovarian stimulation in normogonadotrophic
women with initial inadequate ovarian response torFSH. A multicentre, prospective, randomized controlled trial. Hum Reprod. 2005;20(2):390–6.
67. Ferraretti AP, Gianaroli L, Magli MC, D'angelo A,
Farfalli V, Montanaro N. Exogenous luteinizing hormone in controlled ovarian hyperstimulation forassisted reproduction techniques. Fertil Steril.
2004;82(6):1521–6.
68. Hurwitz JM, Santoro N. Inhibins, activins, and follistatin in the aging female and male. Semin Reprod
Med. 2004;22(3):209–17.
69. Alviggi C, Clarizia R, Pettersson K, et al. Suboptimal
response to GnRHa long protocol is associated witha common LH polymorphism. Reprod Biomed
Online. 2009;18(1):9–14.
70. Piltonen T, Koivunen R, Ruokonen A, Tapanainen
JS. Ovarian age-related responsiveness to humanchorionic gonadotropin. J Clin Endocrinol Metab.
2003;88(7):3327–32.
71. Vihko KK, Kujansuu E, Mörsky P, Huhtaniemi I,
Punnonen R. Gonadotropins and gonadotropin
receptors during the perimenopause. Eur J Endocrinol.
1996;134(3):357–61.
72. Davison SL, Bell R, Donath S, Montalto JG, Davis
SR. Androgen levels in adult females: changes withage, menopause, and oophorectomy. J Clin Endocrinol
Metab. 2005;90(7):3847–53.
73. Marrama P, Montanini V, Celani MF, et al. Decreasein luteinizing hormone biological activity/immunoreactivity ratio in elderly men. Maturitas. 1984;5(4):223–31.
74. Lévy DP, Navarro JM, Schattman GL, Davis OK,
Rosenwaks Z. The role of LH in ovarian stimulation:exogenous LH: let's design the future. Hum Reprod.
2000;15(11):2258–65. Review. Erratum in: Hum
Reprod. 2001;16(3):598.

75. Bosch E, Labarta E, Crespo J, Simón C, Remohí J,
Pellicer A. Impact of luteinizing hormone administration on gonadotropin-releasing hormone antagonist cycles: an age-adjusted analysis. Fertil Steril.
2011;95(3):1031–6.
76. König TE, van der Houwen LE, Overbeek A, Hendriks
ML, Beutler-Beemsterboer SN, Kuchenbecker WK,
et al. Recombinant LH supplementation to a standard
GnRH antagonist protocol in women of 35 years orolder undergoing IVF/ICSI: a randomized controlled
multicentre study. Hum Reprod. 2013;28(10):2804–12.
77. König TE, van der Houwen LE, Lambalk
CB. Recombinant LH supplementation in women of
35 years and older undergoing IVF? Fertil Steril.
2012;98(1):e10–1.
78. Bosch E. Comment on ‘Recombinant LH supplementation to a standard GnRH antagonist protocol inwomen of 35 years old or older undergoing IVF/ICSI: a randomized controlled multicentre study’.
Hum Reprod. 2014;29(3):636–7.
79. Ferraretti AP, La Marca A, Fauser BC, Tarlatzis B,
Nargund G, Gianaroli L, ESHRE Working Group on
Poor Ovarian Response Deﬁnition. ESHRE consensus on the deﬁnition of ‘poor response’ to ovarian
stimulation for in vitro fertilization: the Bologna criteria. Hum Reprod. 2011;26(7):1616–24.
80. Alviggi C, Clarizia R, Pettersson K, et al. Mutation
LH beta subunit is associated with higher exogenous
FSH consumption of FSH during ovarian stimulation. Hum Reprod. 2009;24 Suppl 1:i31.
81. Pettersson K, Söderholm JR. Individual differencesin lutropin immunoreactivity revealed by monoclonal antibodies. Clin Chem. 1991;37(3):333–40.
82. Themmen APN, Huhtaniemi IT. Mutations ofgonadotropins and gonadotropin receptors: elucidating the physiology and pathophysiology of pituitarygonadal function. Endocr Rev. 2000;21(5):551–83.
83. Alviggi C, Pettersson K, Longobardi S, Andersen
CY, Conforti A, De Rosa P, et al. A common polymorphic allele of the LH beta-subunit gene is associated with higher exogenous FSH consumption
during controlled ovarian stimulation for assisted
reproductive technology. Reprod Biol Endocrinol.
2013;11(1):51.
84. Fleming R, Rehka P, Deshpande N, Jamieson ME,
Yates RW, Lyall H. Suppression of LH during ovarian stimulation: effects differ in cycles stimulatedwith puriﬁed urinary FSH and recombinant
FSH. Hum Reprod. 2000;15(7):1440–5.
85. Balasch J, Vidal E, Peñarrubia J, Casamitjana R,
Carmona F, Creus M, et al. Suppression of LH during ovarian stimulation: analysing threshold values
and effects on ovarian response and the outcome ofassisted reproduction in down-regulated women
stimulated with recombinant FSH. Hum Reprod.
2001;16(8):1636–43.
86. Cole LA, Khanlian SA, Muller CY. Detection of
perimenopause or postmenopause human chorionicgonadotropin: an unnecessary source of alarm. Am
J Obstet Gynecol. 2008;198(3):275.e1–7.

s.esteves@androfert.com.br

16

The Role of LH in Controlled Ovarian Stimulation

87. Cole LA, Khanlian SA, Muller CY. Normal productionof human chorionic gonadotropin in perimenopausal
and menopausal women and after oophorectomy. Int J
Gynecol Cancer. 2009;19(9):1556–9.
88. Howles CM. Genetic engineering of human FSH(Gonal-F). Hum Reprod Update. 1996;2(2):172–91.
89. Olijve W, de Boer W, Mulders JW, van Wezenbeek
PM. Molecular biology and biochemistry of humanrecombinant follicle stimulating hormone (Puregon).
Mol Hum Reprod. 1996;2(5):371–82.
90. Practice Committee of American Society for
Reproductive Medicine, Birmingham, Alabama.
Gonadotropin preparations: past, present, and future
perspectives. Fertil Steril. 2008;90(5 Suppl):S13–20.
91. Alviggi C, Revelli A, Anserini P, et al. A prospective,
randomised, controlled clinical study on the assessment of tolerability and of clinical efﬁcacy of
Merional (hMG-IBSA) administered subcutaneously versus Merional administered intramuscularly
in women undergoing multifollicular ovarian stimulation in an ART programme (IVF). Reprod Biol
Endocrinol. 2007;5:45.
92. Platteau P, Laurent E, Albano C, et al. An open, randomized single-centre study to compare the efﬁcacy
and convenience of follitropin beta administered bya pen device with follitropin alpha administered by aconventional syringe in women undergoing ovarian
stimulation
for
IVF/ICSI.
Hum
Reprod.
2003;18(6):1200–4.
93. Bosch E. Recombinant human follicular stimulating
hormone and recombinant human luteinizing hormone in a 2:1 ratio combination. Pharmacologicalcharacteristics and clinical applications. Expert Opin
Biol Ther. 2010;10(6):1001–9.
94. Picard M, Rossier C, Papasouliotis O, Lugan
I. Bioequivalence of recombinant human FSH andrecombinant human LH in a ﬁxed 2:1 combination:
two phase I, randomised, crossover studies. Curr
Med Res Opin. 2008;24(4):1199–208.
95. Gervais A, Hammel YA, Pelloux S, et al.
Glycosylation of human recombinant gonadotrophins: characterization and batch-to-batch consistency. Glycobiology. 2003;13(3):179–89.
96. Hugues JN, Barlow DH, Rosenwaks Z, et al.
Improvement in consistency of response to ovarian
stimulation with recombinant human follicle stimulating hormone resulting from a new method forcalibrating the therapeutic preparation. Reprod
Biomed Online. 2003;6(2):185–90.
97. Giudice E, Crisci C, Eshkol A, Papoian
R. Composition of commercial gonadotrophin preparations extracted from human post-menopausal
urine: characterization of non-gonadotrophin proteins. Hum Reprod. 1994;9(12):2291–9.
98. van de Weijer BH, Mulders JW, Bos ES, VerhaertPD, van den Hooven HW. Compositional analyses ofa human menopausal gonadotrophin preparation
extracted from urine (menotropin). Identiﬁcation ofsome of its major impurities. Reprod Biomed
Online. 2003;7(5):547–57.

195
99. Kuwabara Y, Mine K, Katayama A, Inagawa T, Akira
S, Takeshita T. Proteomic analyses of recombinanthuman follicle-stimulating hormone and urinaryderived gonadotropin preparations. J Reprod Med.
2009;54(8):459–66.
100. Bassett RM, Driebergen R. Continued improvements in the quality and consistency of follitropin
alfa, recombinant human FSH. Reprod Biomed
Online. 2005;10(2):169–77.
101. Driebergen R, Baer G. Quantiﬁcation of follicle
stimulating hormone (follitropin alfa): is in vivo bioassay still relevant in the recombinant age? Curr
Med Res Opin. 2003;19(1):41–6.
102. Steelman SL, Pohley FM. Assay of follicle stimulating hormone based on the augmentation with humanchorionic gonadotropin. Endocrinology. 1953;53(6):604–16.
103. Rozell TG, Okrainetz RJ. FSH: one hormone withmultiple forms, or a family of multiples hormones.
In: Chedrese PJ, editor. Reproductive endocrinology: a molecular approach. New York: Springer
Science + Business Media, LLC; 2009. p. 145–60.
104. Le Cotonnec JY, Porchet HC, Beltrami V, Munafo
A. Clinical pharmacology of recombinant humanluteinizing hormone: Part I. Pharmacokinetics after
intravenous administration to healthy female volunteers and comparison with urinary human luteinizing
hormone. Fertil Steril. 1998;69(2):189–94.
105. Casarini L, Lispi M, Longobardi S, Milosa F, La
Marca A, Tagliasacchi D, et al. LH and hCG action
on the same receptor results in quantitatively andqualitatively different intracellular signalling. PLoS
One. 2012;7(10), e46682.
106. Grzesik P, Teichmann A, Furkert J, Rutz C, Wiesner
B, Kleinau G, et al. Differences between lutropinmediated and choriogonadotropin-mediated receptor
activation. FEBS J. 2014;281(5):1479–92.
107. Karlsson AB, Maizels ET, Flynn MP, Jones JC,
Shelden EA, et al. Luteinizing hormone receptorstimulated progesterone production by preovulatory
granulosa cells requires protein kinase A-dependent
activation/dephosphorylation of the actin dynamizing
protein coﬁlin. Mol Endocrinol. 2010;24(9):1765–81.
108. Chin EC, Abayasekara DRE. Progesterone secretion byluteinizing human granulosa cells: a possible cAMPdependent but PKA-independent mechanism involved
in its regulation. J Endocrinol. 2004;183(1):51–60.
109. Ryan KE, Glister C, Lonergan P, Martin F, Knight
PG, et al. Functional signiﬁcance of the signal transduction pathways Akt and Erk in ovarian follicles:in vitro and in vivo studies in cattle and sheep. J
Ovarian Res. 2008;1(1):2.
110. Noma N, Kawashima I, Fan H-Y, Fujita Y, Kawai T,
et al. LH-induced neuregulin 1 (NRG1) type III transcripts control granulosa cell differentiation and oocyte
maturation. Mol Endocrinol. 2011;25(1):104–16.
111. Sekiguchi T, Mizutani T, Yamada K, Kajitani T,
Yazawa T, et al. Expression of epiregulin and amphiregulin in the rat ovary. J Mol Endocrinol. 2004;
33(1):281–91.

s.esteves@androfert.com.brS.C. Esteves and C. Alviggi

196
112. Choi J-H, Chen C-L, Poon SL, Wang H-S, Leung
PCK. Gonadotropin-stimulated epidermal growth
factor receptor expression in human ovarian surface
epithelial cells: involvement of cyclic AMPdependent exchange protein activated by cAMP
pathway. Endocr Relat Cancer. 2009;16(1):179–88.
113. Yamashita Y, Hishinuma M, Shimada M. Activationof PKA, p38 MAPK and ERK1/2 by gonadotropinsin cumulus cells is critical for induction of EGF-likefactor and TACE/ADAM17 gene expression duringin vitro maturation of porcine COCs. J Ovarian Res.
2009;2:20.
114. Ulloa-Aguirre A, Crépieux P, Poupon A, Maurel
M-C, Reiter E. Novel pathways in gonadotropin
receptor signalling and biased agonism. Rev Endocr
Metab Disord. 2011;12(4):259–74.
115. Schiffer Z, Keren-Tal I, Deutsch M, Dantes A, Aharoni
D, et al. Fourier analysis of differential light scattering
for the quantitation of FSH response associated withstructural changes in immortalized granulosa cells.
Mol Cell Endocrinol. 1996;118(1–2):145–53.
116. Keren-Tal I, Suh BS, Dantes A, Lindner S, Oren M,
et al. Involvement of p53 expression in cAMPmediated apoptosis in immortalized granulosa cells.
Exp Cell Res. 1995;218(1):283–95.
117. Amsterdam A, Dantes A, Liscovitch M. Role ofphospholipase-D and phosphatidic acid in mediating
gonadotropin-releasing hormone-induced inhibitionof preantral granulosa cell differentiation.
Endocrinology. 1994;135(3):1205–11.
118. Aharoni D, Dantes A, Oren M, Amsterdam A.
cAMP-mediated signals as determinants for apoptosis in primary granulosa cells. Exp Cell Res.
1995;218(1):271–82.
119. Sun Q-Y, Miao Y-L, Schatten H. Towards a newunderstanding on the regulation of mammalianoocyte meiosis resumption. Cell Cycle. 2009;8(17):2741–7.
120. Johnson AL, Bridgham JT, Swenson JA. Activationof the Akt/protein kinase B signalling pathway is
associated with granulosa cell survival. Biol Reprod.
2001;64(5):1566–74.
121. Peter AT, Dhanasekaran N. Apoptosis of granulosa
cells: a review on the role of MAPK-signalling modules. Reprod Domest Anim. 2003;38(3):209–13.
122. Craig J, Orisaka M, Wang H, Orisaka S, Thompson
W, et al. Gonadotropin and intra-ovarian signals
regulating follicle development and atresia: the delicate balance between life and death. Front Biosci.
2007;12:3628–39.
123. Grøndahl ML, Borup R, Lee YB, Myrhøj V, Meinertz
H, Sørensen S. Differences in gene expression of
granulosa cells from women undergoing controlled
ovarian hyperstimulation with either recombinantfollicle-stimulating hormone or highly puriﬁed

human menopausal gonadotropin. Fertil Steril.
2009;91(5):1820–30.
124. Menon KM, Munshi UM, Clouser CL, Nair
AK. Regulation of luteinizing hormone/human chorionic gonadotropin receptor expression: a perspective. Biol Reprod. 2004;70(4):861–6.
125. Bosch E, Vidal C, Labarta E, Simon C, Remohi J,
Pellicer A. Highly puriﬁed hMG versus recombinant
FSH in ovarian hyperstimulation with GnRH antagonists-a randomized study. Hum Reprod. 2008;23(10):2346–51.
126. Ruvolo G, Bosco L, Pane A, Morici G, Cittadini E,
Roccheri MC. Lower apoptosis rate in human cumulus cells after administration of recombinant luteinizing hormone to women undergoing ovarian
stimulation for in vitro fertilization procedures.
Fertil Steril. 2007;87(3):542–6.
127. Carone D, Caropreso C, Vitti A, Chiappetta
R. Efﬁcacy of different gonadotropin combinations
to support ovulation induction in WHO type I anovulation infertility: clinical evidences of human recombinant FSH/human recombinant LH in a 2:1 ratio
and highly puriﬁed human menopausal gonadotropin stimulation protocols. J Endocrinol Invest.
2012;35(11):996–1002.
128. Pacchiarotti A, Sbracia M, Frega A, Selman H,
Rinaldi L, Pacchiarotti A. Urinary hMG (Menopur)
versus recombinant FSH plus recombinant LH(Pergoveris) in IVF: a multicenter, prospective, randomized controlled trial. Fertil Steril. 2010;
94(6):2467–9.
129. Bühler KF, Fischer R. Recombinant human LH supplementation versus supplementation with urinary
hCG-based LH activity during controlled ovarian
stimulation in the long GnRH-agonist protocol: amatched case-control study. Gynecol Endocrinol.
2012;28(5):345–50.
130. Fábregues F, Creus M, Casals G, Carmona F, BalaschJ. Outcome from consecutive ICSI cycles in patientstreated with recombinant human LH and those supplemented with urinary hCG-based LH activity during controlled ovarian stimulation in the long
GnRH-agonist protocol. Gynecol Endocrinol. 2013;29(5):430–5.
131. Bühler K, Naether OG, Bilger W. A large, multicentre, observational, post-marketing surveillance studyof the 2:1 formulation of follitropin alfa and lutropin
alfa in routine clinical practice for assisted reproductive technology. Reprod Biol Endocrinol. 2014;12:6.
132. Papaleo E, Alviggi C, Colombo GL, Pisanelli C,
Ripellino C, Longobardi S, Canonico PL. Costeffectiveness analysis on the use of rFSH + rLH for
the treatment of anovulation in hypogonadotropic
hypogonadal women. Ther Clin Risk Manag.
2014;10:479–84.

s.esteves@androfert.com.br