GnRH Agonist vs.

hCG for Triggering of Ovulation

Differential Effects on Gene Expression in Human
Granulosa Cells
Jigal Haas*., Libby Ophir., Eran Barzilay, Gil M. Yerushalmi, Yuval Yung, Alon Kedem, Ettie Maman,
Ariel Hourvitz
Human Reproduction Lab and IVF Unit, Department of Obstetrics and Gynecology, Chaim Sheba Medical Center, Tel-Hashomer, Ramat Gan, Israel

Abstract
Objective: To investigate the mRNA expression of genes related to steroidogenesis and OHSS in granulosa cells (GCs) of
patients triggered with GnRH agonist compared to patients triggered with hCG.
Design: Mural GCs were obtained at the time of oocyte retrieval and gene expression was analyzed using quantitative real
time RT-PCR.
Settings: Single center, case control study.
Patient(s): 24 women who were treated with GnRH agonist or hCG for triggering of ovulation.
Interventions: GC collection.
Main Outcome Measure(s): The expression of genes related to steroidogenesis and OHSS in mural GCs
Results: The fertilization rate was similar in the two groups. The mRNA expression of CYP19A1 (0.50 vs 1, arbitrary unit),
CYP11A1 (0.6 vs. 1) and 3 beta hydroxysteroid-dehydrogenase (0.39 vs 1) was significantly lower in the GnRH group. The
expression of VEGF (0.74 vs. 1) and inhibin b B (0.38 vs 1) was lower in the GnRH analog triggered group.
Conclusion: Expression of genes related to steroidogenesis is lower at the time of oocyte retrieval in patients triggered with
GnRH agonist. The decreased expression of VEGF and inhibin b B in the GnRH agonist group can explain the mechanism of
early OHSS prevention.
Citation: Haas J, Ophir L, Barzilay E, Yerushalmi GM, Yung Y, et al. (2014) GnRH Agonist vs. hCG for Triggering of Ovulation Differential Effects on Gene
Expression in Human Granulosa Cells. PLoS ONE 9(3): e90359. doi:10.1371/[Link].0090359
Editor: Ana Claudia Zenclussen, Medical Faculty, Otto-von-Guericke University Magdeburg, Medical Faculty, Germany
Received October 26, 2013; Accepted January 29, 2014; Published March 6, 2014
Copyright: 2014 Haas et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors have no support or funding to report.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: jigalh@[Link]
. These authors contributed equally to this work.

growth factor (VEGF) which is known among other proinflammatory cytokines to play a fundamental role in the pathophysiology of OHSS [6,7].
Both inhibinA and inhibin B have been shown to be higher in
patients developing OHSS. The level of inhibin B increase even
before hCG administration and may serve as a predictor of
OHSS, while inhibin A increases after developing OHSS and may
be used to monitor the disease [8]
The LH surge plays a crucial role in the formation of the corpus
luteum. A range of EGF-like ligands including AREG, EREG and
b-cellulin function as LH target genes [9], and AREG is acting as a
growth factor with an EGF-like motif that initiates the morphological and biochemical events triggered by the LH including
cumulus expansion and oocyte maturation[10]. In addition,
Follicle-Stimulating Hormone (FSH) and Luteinizing Hormone
have a central role in follicle growth and maturation through

Introduction
Several studies have shown that the use of GnRH agonist for
triggering of final oocyte maturation prevents the development of
ovarian hyperstimulaion syndrome (OHSS), even in patients at
high risk of developing OHSS [13]. In contrast to hCG, the
GnRH agonist-induced surge resembles the natural mid cycle
surge of gonadotropins and exposes follicles to both LH and FSH.
However, after GnRH agonist triggering the mid cycle surge of
gonadotropins is shorter in duration and amplitude compared with
the natural cycle [4,5]. The precise mechanism of OHSS
prevention is not thoroughly understood. One of the hypotheses
is that the shorter half-life of the endogenous LH surge induced by
GnRH agonist, compared with the continuous high levels of hCG
stimulating the LH receptor, induces a shorter and milder
secretion of vasoactive substances such as vascular endothelial

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GnRH Agonist Triggering Compared to hCG

performed by transvaginal ultrasound-guided needle aspiration,

only follicles $16 mm were retrieved.

interaction with their receptors, the Follicle Stimulating Hormone

Receptor (FSHR) and Luteinizing Hormone Choriogonadotropin
Receptor (LHCGR).
Although previous described that more mature oocytes were
retrieved by the use of GnRH agonist for ovulation triggering [11],
a poor clinical outcome with high early pregnancy loss rate was
reported in this group of pregnancies [1113]. These results can be
explained by the luteal phase insufficiency caused by lysis of the
corpus luteum which results in lower levels of progesterone in the
group of women triggered with GnRh agonist. It is well established
that progesterone formation depends on the activation of
steroidogenesis genes such as STAR, which uptakes the cholesterol
into the mitochondria, and CYP11A1, that converts cholesterol to
pregnenolone. CYP19A1 converts androstendione to estradiol
which is crucial for endometrium formation. Therefore, these
steroidogenesis genes play a crucial role in the luteal phase and in
early pregnancy stages.
The aim of this study was to investigate the mRNA expression
of genes related to steroidogenesis and OHSS as well as to
investigate other genes such as LH receptor (LHCGR), FSH
receptor (FSHR), amphiregulin and epiregulin, at the early stage
of 36 hours post triggering, in granulosa cells of patients triggered
with GnRH agonist compared to patients triggered with hCG.

Mural Granulosa Cell Purification

Mural GCs were collected from pooled follicular fluid [14],
avoiding blood clots, and resuspended in phosphate-buffered
solution (PBS). After allowing the cells to settle by gravity for a few
minutes, the top medium was aspirated. This step was repeated
two to three times until the medium was clear. The cells were
centrifuged at 200 g for 5 minutes at room temperature, and the
resulting pellets were resuspended in red blood cell (RBC) lysis
buffer (Sigma-Aldrich). After a 15-minutes incubation period at
37uC, the cells were centrifuged at 200 g for 5 minutes at room
temperature, and the resulting pellets were subjected to RNA
purification.
The method we used for the isolation of the granulosa cells has
been used in previous studies by us and by other authors [1518].

RNA Extraction and qRT-PCR

Total RNA was extracted from GCs by a Mini RNA Isolation I
Kit (Zymo Research Corp.), according to the manufacturers
instructions. One microliter of RNA solution was used for reverse
transcription with a high-capacity cDNA RT kit (Applied
Biosystems) according to the manufacturers instructions. A power
SYBR Green PCR mix (Applied Biosystems) was used for the
PCR step. Amplification and detection were performed using the
StepOnePlus real-time PCR system (Applied Biosystems) with the
following profile: 1 cycle at 95uC for 20 seconds, 40 cycles each at
95uC for 3 seconds, and 60uC for 30 seconds. One microgram of
complementary DNA (cDNA) was used per reaction in a 10-mL
reaction volume. All samples were run in duplicates. The b-actin
RNA was chosen as a suitable normalization control gene. The
same quantitative real-time PCR protocol was used for all the
genes analyzed. Results are expressed as fold change with respect
to the experimental control. For primers details, see Table S1.
In order to verify that the white blood cells (WBC) were
removed, we measured the mRNA expression of CD45 (which
identify WBCs) in the GCs and found negligible expression of CD
45 which demonstrated that the WBCs were removed from the
samples.

Materials and Methods

The research was approved by the authors institutional review
board at Tel Hashomer hospital, IRB number 8707-11-SMC and
written informed consent was obtained from each participating
subject

GnRH agonist or hCG triggering

The IVF patients included in the study were women ,40 years
of age undergoing IVF due to male factor infertility, tubal factor,
or for the purpose of preimplantation genetic diagnosis (PGD,
genetic tests of embryos prior implantation). Patients with
infertility related to female pathology, such as poor ovarian
response and endometriosis were excluded from the study.
Intracytoplasmic sperm injection (ICSI) was performed only in
cases of male factor infertility. All decisions regarding treatment
protocol including mode of triggering were made according to
physician preference. The treating physicians in the clinic were
acting independently so that decisions regarding IVF protocol
were taken impartially.
During the study period all patients who met the inclusion
criteria sited above and were selected to receive agonist triggering
by the treating physician according to his decision were recruited
to the study. All 12 patients gave their consent and were recruited
to the study. For the control group we recruited 12 consenting
patients triggered with hCG while matching according to age,
etiology of infertility, total dose of gonadotropins used for ovarian
stimulation and length of treatment.
The granulosa cells (GCs) were obtained at the time of oocyte
retrieval for IVF procedures. The women were treated using the
antagonist protocol with GnRH antagonist (Cetrorelix; Merck
Serono or Orgalutran; Schering-Plough). Ovarian stimulation
with a daily SC dose of recombinant FSH (Gonal-F, MerckSerono or Puregon Pen, Schering-Plough) and hMG (Menogon;
Ferring) was started on the third day of the menstrual cycle. The
amount of the initial dose depended on the age, body mass index
(BMI), and treatment history. When three leading follicles reached
18 mm in diameter, the women received GnRH agonist
(Decapeptyl 0.2 mg; Ferring) or hCG (Ovitrelle 250 mg; Merck
Serono) for final oocyte maturation. Oocyte retrieval was
PLOS ONE | [Link]

E2 measurement
The serum was analyzed for E2 concentration at the day of
triggering using an E2 ELISA kit (Enzo Life Sciences Int,
Farmingdale, NY) according to the manufacturers instructions
(Sensitivity 14.0 pg/ml (range 15.61,000 pg/ml)).

Statistical analysis
Comparisons were performed using the unpaired two-tailed
students t-test assuming a normal distribution with unequal
variances. A P#0.05 was considered statistically significant.

Results
There was no difference between the patients in the two groups
in terms of age, duration of ovulation stimulation and the total
dose of gonadotropins required for ovarian stimulation. In the
group triggered with GnRH agonist, the average estradiol level at
the day of ovulation triggering was significantly higher than in the
group triggered with hCG (11853 pmol/L vs 8619 pmol/L, P,
0.01). The number of eggs retrieved was similar in the two groups
(13.75 vs 11.72 p = 0.35), the fertilization rate was similar in the
two groups (Table 1). The women in the study group were at
moderate risk of developing OHSS and in the control group, the
2

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GnRH Agonist Triggering Compared to hCG

Table 1. Characteristics of the patients and IVF procedures included in this study.

Triggering with GnRH agonist

Triggering with Hcg

p value

12

Age (mean)

31.8

3.3

31.7

12
6

4.8

NS
Ns

E2 (mean)

11853

2075.5

8619

2757.3

0.006

oocytes retrieved

13.75

5.8

11.72

3.2

NS

duration of stimulation

11.58

1.8

11.2

1.7

NS

% fertilization

70

0.2

54

0.2

0.07

doi:10.1371/[Link].0090359.t001

fluid was similar in the two groups. Epiregulin expression was

higher in the GnRH triggered group compared to the hCG
triggered group albeit with no statistical significance (1.92 vs 1,
P = 0.17) (Figure 11).
To determine whether a link exists between the levels of
estradiol and the mRNA expression of the different genes
(amphiregulin, epiregulin, VEGF, LHCGR, FSHR) we divided
the patients according to the serum estradiol levels on the day of
triggering and according to the way of triggering. The patients
with the highest (n = 4) and with the lowest(n = 4) levels of
estradiol, in the group triggered with hCG were divided to two
groups. One group (n = 4) consisted of patients with lower levels of
estradiol (44005960 pmol/L) while the other group (n = 4)
consisted of patients with higher levels of estradiol (11000
12050 pmol/L) on the day of triggering. We did not find any
differences of expression between the two groups regarding all the
genes that were evaluated. Patients triggered with GnRH agonist
were divided to two groups at the same way. One group (n = 4)
consisted of patients with lower levels of estradiol (8410
10700 pmol/L) while the other group (n = 4) consisted of patients
with higher levels of estradiol (1381015970 pmol/L) on the day
of triggering. In this group also, we did not find any differences of

risk for OHSS was lower demonstrated by lower levels of estrogen

at the day of triggering.
VEGF mRNA expression was lower in GCs retrieved from
women triggered with GnRH agonist compared to women
triggered with hCG (0.74 vs 1, arbitrary unit, P = 0.05). Expression
of inhibin b B was significantly lower in the GnRH agonist
triggered group (0.38 vs 1, P = 0.01). We did not find any
differences between the two groups regarding mRNA expression
of inhibin a (Figures 13).
We examined the mRNA expression of different enzymes
involved in steroidogenesis (Figures 47). The mRNA expression
of CYP19A1 (0.50 vs 1, P,0.01), CYP11A1 (0.6 vs 1, P = 0.02)
and 3 beta-hydroxysteroid-dehydrogenase (0.39 vs 1, P = 0.03) was
significantly lower in the GnRH agonist group compared to the
hCG group. Expression of STAR was similar in the two groups.
We found no difference in expression of LHCGR between the
two groups (Figure 8). FSHR mRNA expression was significantly
lower in the GnRH triggered group compared to the hCG
triggered group (0.4 vs 1, P,0.01) (Figure 9). Amphiregulin
expression was significantly higher in the GnRH triggered group
compared to the hCG triggered group (2.32 vs 1, P,0.01)
(Figure 10). Despite the significant difference in amphiregulin
expression in mural GCs, the level of amphiregulin in the follicular

Figure 1. Expression of VEGF in mGCs obtained from IVF cycles separated according to ovulation triggering.
doi:10.1371/[Link].0090359.g001

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Figure 2. Expression of Inhibin a in mGCs obtained from IVF cycles separated according to ovulation triggering.
doi:10.1371/[Link].0090359.g002

expression between the two groups regarding all the genes that
were evaluated.

CYP11 and 3 beta-hydroxysteroid-dehydrogenase, and expression

of FSHR and amphiregulin.

Discussion

Prevention of OHSS

To the best of our knowledge, this is the first study to

systematically explore possible differences in mRNA expression of
enzymes and receptors in granulosa cells of patients triggered with
GnRH agonist compared to patients triggered with hCG.
This study demonstrates that the mode of ovulation triggering
significantly affects mRNA expression of proteins that have been
suggested to participate in the development of OHSS, such as
VEGF and inhibin beta, as well as expression of important
enzymes in the process of steroidogenesis, such as CYP19A1,

All studies published to date have shown that the use of GnRH
agonist for triggering of ovulation prevents the development of
ovarian hyperstimulaion syndrome even in patients which are at
high risk of developing OHSS. The precise mechanism for
preventing OHSS is not thoroughly understood.
Early studies have shown that VEGF might be responsible for
the increase in vascular permeability and ascites development in
women developing OHSS [7,19]. Serum VEGF have been
reported to be significantly higher in patients who developed
severe OHSS than in patients at risk for OHSS who did not

Figure 3. Expression of Inhibin b B in mGCs obtained from IVF cycles separated according to ovulation triggering.
doi:10.1371/[Link].0090359.g003

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GnRH Agonist Triggering Compared to hCG

Figure 4. Expression of CYP19A1 in mGCs obtained from IVF cycles separated according to ovulation triggering.
doi:10.1371/[Link].0090359.g004

develop the syndrome [20], while other studies [21,22] did not
demonstrate elevated levels of VEGF in patients developing
OHSS.
Previous studies comparing blood serum VEGF levels in women
triggered with GnRH agonist to women triggered with hCG did
not find any differences between the two groups [23,24].
Few studies measured follicular fluid levels of VEGF in women
triggered with GnRH agonist compared to women triggered with
hCG. Some of the researchers found lower levels of VEGF in the
group of women triggered with GnRH agonist [24,25] while
others did not find a difference between the two groups [26].

In our study we measured mRNA expression of VEGF and

found it to be lower with borderline significance in the group who
received GnRH agonist for final triggering. Our findings correlates
with previous findings of Cerillo et al[25], that demonstrated
significantly lower levels of VEGF mRNA expression in granulose
cells obtained from women triggered with GnRH agonist
compared with women triggered with hCG.
The lower levels of VEGF, which is known, among other
proinflammatory cytokines, to play a fundamental role in the
pathophysiology of OHSS, may explain why OHSS is prevented
among women triggered with GnRH agonist. The lower VEGF

Figure 5. Expression of STAR in mGCs obtained from IVF cycles separated according to ovulation triggering.
doi:10.1371/[Link].0090359.g005

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Figure 6. Expression of CYP11A1 in mGCs obtained from IVF cycles separated according to ovulation triggering.
doi:10.1371/[Link].0090359.g006

rat model [27], and mRNA expression of inhibin beta B which

expresses the presence of inhibin B.
As expected, we found the expression of inhibin b B to be
significantly lower in the GnRH analog triggered group (0.38 vs 1,
P = 0.01). The lower levels of inhibin b B among women triggered
with GnRH agonist may play a role in prevention of OHSS.

mRNA expression may reflect the short LH signaling in women

triggered with GnRH agonist.
Innhibin A consists of the alpha subunit and the A type of the
beta unit, while inhibin B consists of the alpha subunit and the B
type of the beta unit. The amino acid sequences of the beta
subunits show 70% homology. The level of both inhibins was
shown to be higher in patients developing OHSS. The level of
inhibin B increase even before hCG administration and may serve
as a predictor of OHSS, while inhibin A increases after developing
OHSS and may be used to monitor the disease [8].
In our study we investigated mRNA expression of inhibin alpha,
which was shown previously to be down regulated in OHSS in a

Lower steroidogenesis
The LH surge plays a crucial role in the formation of the corpus
luteum. As mentioned before after triggering with GnRH agonist,
there is a shorter duration of the LH surge compared to triggering
with hCG, leading to a reduced LH support for the developing
corpus luteum, which may cause early luteolysis. It has been

Figure 7. Expression of 3bHSD in mGCs obtained from IVF cycles separated according to ovulation triggering.
doi:10.1371/[Link].0090359.g007

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GnRH Agonist Triggering Compared to hCG

Figure 8. Expression of LHCGR in mGCs obtained from IVF cycles separated according to ovulation triggering.
doi:10.1371/[Link].0090359.g008

signs of apoptosis, a cascade of cellular events that will eventually

culminate in apoptosis has already begun. We propose that the
different gene expression we found at this early stage might be one
of the first signs of apoptosis.
Despite the apparent early lower levels of steroidogenesis, it is
well known that rescue of the corpus luteum can be achieved by
injecting a small dose of hCG at the day of the ovum pickup
[28,31]. The fact that corpus luteum rescue is possible may imply
that the early luteal dysfunction in the GnRH agonist triggered
group as demonstrated in this study is reversible.

suggested that luteolysis results in significantly lower levels of

estradiol and progesterone after GnRH agonist triggering compared to triggering with hCG [23,28,29]. In our study we wanted
to investigate how early the impairment of the steroidogenesis
begins. We found differences in expression between the two groups
as early as 36 hours after ovulation triggering, at the time of ovum
pickup. Expression of CYP19A1, CYP11A1 and 3 beta-hydroxysteroid-dehydrogenase was significantly lower in the GnRH group
compared to the hCG group. Engmann et al [30], showed that the
granulose cells after triggering with GnRH agonist are still viable
on the day of retrieval and that the rate of apoptosis of granulosa
cells was comparable between the two groups. It is possible that at
this early stage (Oocyte Pick Up), even though there are no overt

Figure 9. Expression of FSHR in mGCs obtained from IVF cycles separated according to ovulation triggering.
doi:10.1371/[Link].0090359.g009

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GnRH Agonist Triggering Compared to hCG

Figure 10. Expression of amphiregulin in mGCs obtained from IVF cycles separated according to ovulation triggering.
doi:10.1371/[Link].0090359.g010

of GCs induces up regulation of amphiregulin and epiregulin. It

was previously suggested that they mediate the LH signal and
partially take a part in the process of cumulus expansion and
oocyte maturation [9]. The concentration of amphiregulin in the
follicular fluid has been shown to correlate inversely with the
fertilization rate whereas little significant association was observed
between the level of amphiregulin and embryo quality [32]. In
contrast, Ben Ami et al. [33] demonstrated that enrichment of
maturation medium with amphiregulin and epiregulin significantly
improved the maturation rate of human Germinal Vesicle oocytes
in vitro. Humaidan et al. measured the levels of amphiregulin in the
follicular fluid after triggering of final oocyte maturation with

LH and FSH receptors

In our study, LHCGR expression was similar in both the
GnRH triggered group and the hCG triggered group, but FSHR
expression was significantly lower in the GnRH triggered group
compared to the hCG triggered group (0.4 vs 1, P,0.01). We
suggest that the supraphysiologic levels of FSH which occurs after
triggering with GnRH agonist may results in negative feedback
which culminates in lower levels of FSH receptors.

Amphiregulin
Amphiregulin and epiregulin are ligands of the epidermal
growth factor receptor, released from mural GCs. LH stimulation

Figure 11. Expression of epiregulin in mGCs obtained from IVF cycles separated according to ovulation triggering.
doi:10.1371/[Link].0090359.g011

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GnRH Agonist Triggering Compared to hCG

either GnRH agonist or hCG. Significantly lower levels of

amphiregulin were found in FF from the GnRH agonist group
as compared to the hCG group. Amphiregulin concentration in
FF was negatively correlated with the fertilization rate. Moreover,
a trend for an inverse correlation was found for amphiregulin and
embryo quality[26].
In our study amphiregulin expression was significantly higher in
GCs retrieved from women who were triggered with GnRH
agonist compared with women triggered with hCG (2.32 vs 1, P,
0.01). We did not find any difference in the levels of amphiregulin
in the FFs. Combining the two groups of women, regardless of the
route of triggering, we found a positive correlation between levels
of mRNA expression of amphiregulin and fertilization rate. The
significantly higher mRNA expression of amphiregulin in the
GnRH agonist triggered group is in a distinct contrast to
previously published data regarding the relations between
amphiregulin, fertilization rate, embryo quality and mode of
ovulation triggering. However, this discrepancy may be explained
by the fact that in our study we examined mRNA expression while
other studies examined amphiregulin levels in the FF. It is well
known that downregulation of EGF receptor signaling occurs
through trafficking of the receptor-ligand complex to lysosomes,
culminating in proteolytic destruction of both the receptor and
ligand [34,35]. Receptor activation may therefore induce uptake
and degradation of amphiregulin, which, in a confined space such
as in the follicle, may induce a significant decrease in amphiregulin
levels. Thus, our findings do not contradict previous published
data since increased production of amphiregulin is not directly
linked to increased levels of the protein in the FF. Based on our
findings regarding expression of amphiregulin and the findings of
Ben Ami et al. regarding addition of amphiregulin to maturation
medium, we suggest that amphiregulin is important to oocyte
maturation and that the contrasting data regarding amphiregulin
levels in FF may be due to ligand uptake and degradation by
cumulus cells.
Like all other non randomized studies there might be a selection
bias in our study. Therefore we matched the hCG triggered group
to the GnRH agonist triggered group according to age, etiology of
infertility, total dose of gonadotropins used for ovarian stimulation

and length of treatment. In the results section we described the

basic parameters of the women in the two groups and as shown
except of the E2 levels, all the other parameters were similar
proving the efficiency of the matching technique. To deal with the
higher E2 levels in the GnRH agonist group we divided the
patients according to the serum E2 levels on the day of triggering
and compared the expression profile of the genes. We did not find
any differences of expression between the two groups regarding all
the genes that were evaluated.
However, we cant rule out completely any selection bias and
therefore farther study with patient randomization should be
performed to strengthen our results.
In conclusion, our findings suggest that the decreased expression
of VEGF and inhibin b B in the GnRH agonist group can explain
the mechanism of early OHSS prevention in these women. The
expression of enzymes which take part in steroidogenesis of
estrogen and progesterone is lower at the time of oocyte retrieval
in patients triggered with GnRH agonist. Taken together, we have
shown that gene expression in granulosa cells is strongly linked to
the method of ovulation triggering. The fact that gene expression
pattern is profoundly different between hCG and GnRH agonist
triggered women raises the possibility that granulosa function may
also be linked to ovulation triggering method. The cellular and
clinical effects of these differences of expression as well as the
possible effect on oocyte function and quality remains to be further
studied.

Supporting Information
Table S1 Real time polymerase chain reaction primers

sequence.
(DOCX)

Author Contributions
Conceived and designed the experiments: JH LO YY AK AH. Performed
the experiments: JH LO EB YY. Analyzed the data: JH LO EM.
Contributed reagents/materials/analysis tools: LO GY. Wrote the paper:
JH EB GY AK.

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