Open Journal of Obstetrics and Gynecology, 2011, 1, 31-35
doi:10.4236/ojog.2011.12007 Published Online June 2011 (http://www.SciRP.org/journal/ojog/ OJOG ).
Published Online June 2011 in SciRes. http://www.scirp.org/journal/OJOG
Recombinant human luteinizing hormone supplementation
may improve embryo quality in in vitro fertilization/
intracytoplasmic sperm injection cycles with
gonadotropin-releasing hormone antagonist protocol
Amir Wiser, Ariel Hourvitz, Yo av Y inon, Jacob Levron, Jehoshua Dor, Shai Elazar Elizr u
IVF Unit, Division of Obstetrics and Gynecology, Chaim Sheba Medical Center, Tel Hashomer Hospital, Affiliated with the Sackler
School of Medicine, Tel Aviv University, Tel Aviv, Israel.
Received 18 March 2011; revised 16 April 2011; accepted 25 April 2011.
ABSTRACT
Objective: To evaluate the effects of recombinant
LH (rLH) supplementation on embryo quality in
IVF/ICSI cycles with GnRH antagonist. Study de-
sign: Prospective, randomized controlled study.
Thirty women were enrolled, 15 in the study (FSH
+ rLH) group and 15 in the control (rFSH only)
group. On the day GnRH antagonist was started,
the study group patients received 75 IU of rLH in
addition to rFSH. The main outcome measures
were embryo quality, number of oocytes retrieved,
and fertilization rate. Results: The rLH group had
significantly more top-quality embryos (36/43, 84%)
compared to the control group (40/68, 59%; p =
0.006). Fertilization rates and number of oocytes
retrieved were similar between groups. Progester-
one and estradiol (E2) concentrations in follicular
fluid were higher in the study group compared to
controls (16.5 ± 2.5 µg/ml vs. 11.4 ± 3.6 µg/ml pro-
gesterone, P = 0.07; and 687 ± 112 pg/ml vs. 471 ±
65 pg/ml E2, p = 0.08). Conclusion: Adding rLH to
ovarian stimulation with GnRH antagonist can
yield higher quality embryos.
Keywords: Recombinants LH; Antagonist Protocol;
Embryo Quality; IVF Treatment
1. INTRODUCTION
Despite the vast experience in various in vitro fertiliza-
tion (IVF) protocols, the beneficial effect of LH supple-
mentation during ovarian stimulation is still far from
clear [1]. This controversy is supported by the theory of
“LH ceiling levels,” a yet-to-be-defined serum LH con-
centration, above which LH is believed to cause detri-
mental effects on oocyte development and implantation
[2].
Gonadotropin-releasing hormone (GnRH) antagonists
offer the opportunity to control endogenous LH rapidly.
GnRH antagonists act on gonadotroph secretion cells
through the immediate, competitive blockade of GnRH
receptors and induce a marked decrease in serum LH
levels and a less pronounced decrease in FSH secretion
[3]. Data regarding the role of LH activity in GnRH an-
tagonist protocols are scarce and firm conclusions can-
not be drawn [4]. The MERIT study [5] has been sug-
gested that supplementation ovarian stimulation with LH
during long GnRH agonist protocol has a beneficial ef-
fect on embryo quality. However, very few studies re-
ported on recombinant LH (rLH) addition to GnRh an-
tagonist cycles [6,7]. One study [7] found no evident
benefit to rLH supplementation during GnRH antagonist
cycle. On the other hand, Acevedo et al. [6] described
that rLH supplementation improved the rate of top qual-
ity embryos in recipients whose embryos originate from
GnRH- antagonist-treated donors.
Therefore, the aim of this prospective study was to
evaluate the effects of rLH supplementation on embryo
quality in IVF/ICSI cycles using a GnRH antagonist
protocol.
2. MATERIAL AND METHODS
A prospective, randomized, controlled study was de-
signed. Inclusion criteria were women with a normal
menstrual cycle (25 - 34 days), 20 to 36 years of age,
and BMI less than 30 kg/m2. Exclusion criteria included
polycystic ovaries, basal FSH > 10 IU/Ml and more than
five previous IVF failures. The study was approved by
the local Institutional Review Board and written in-
formed consent was obtained from each participant after
detailed explanation.

A. W iser et al. / Open Journal of Obstetrics and Gynecology 1 (2011) 31-35 32
On day three of the menstrual cycle, all patients un-
derwent ultraso und to exclude ovarian cysts and a blood
test for serum estradiol (E2), progesterone and LH levels.
When no ovarian cysts were observed, E2 level was be-
low 40 pg/ml and progesterone level below 1.0 ng/ml,
ovarian stimulation was started. The patients were ran-
domized, during the initial visit in the clinic, to rLH 75
IU supplementation at the day when GnRH antagonist
was started (study group) and rFSH only (control group).
The two groups started the ovarian stimulation with 150
- 225 IU/day rFSH (Gonal F, Merck, Serono SA, Au-
bunne, Switzerland) for five days. After five days, the
patients underwent ultrasound and blood test for E2,
progesterone, and LH levels; rFSH dose was adjusted
when necessary. When the leading follicle reached 13
mm in diameter, GnRH antagonist (Cetrotide Merck,
Serono SA, Switzerland) was added. Study group pa-
tients received 75 IU of rLH (Luveris, Merck, Serono
SA, Switzerland) starting the same day as GnRH an-
tagonist, combined with the rFSH dose, whereas those
randomized to the control group continued stimulation
with rFSH only, during the entire ovarian stimulation
period. When at least three leading follicles achieved an
18 mm diameter, 250 mcg of recombinant hCG (Ovitrel,
Merck Serono SA, Bari, Italy) was administered. Ovum
pick up was performed 36 hours later. On the day of
OPU, four leading follicles were aspirated separately. E2,
progesterone, FSH, and LH levels in the follicular fluid
were measured after removal of the oocytes. Fertilization
was assessed 20 hours after insemination for the ap-
pearance of two pronuclei. Embryos were graded from
one to four, based on fragmentation rate and the size and
number of blastomers: grade 4 embryos were equal-
sized symmetrical blastomers with no fragmentation;
grade 3 were equal-sized symmetrical cells with less
than 10% fragmentation; grade 2 were non-symmetrical
blastomers with 10% - 50% fragmentation; and grade 1
had more than 50% fragmentation. Embryos graded 3
and 4 were transferred and the remaining embryos were
cryopreserved. Up to three, best-quality embryos were
transferred on day two or three, (according to Israeli
Fertility Association policy guidelines) and the remain-
ing top-quality embryos were cryopreserved.
The following parameters were compared between the
two groups serum E2, progesterone, and LH concentra-
tions (at the time of hCG administration) , follicular fluid
levels of E2, progesterone, FSH, and LH, the number of
retrieved oocytes, fertilization rate, embryo quality, and
pregnancy rate. The main outcome measure was embryo
quality.
Statistical analysis: The chi-square and Fisher’s exact
tests were used to compare proportions. Continuous va-
riables (presented as mean and SD) tested by student
t-test or ANOVA and p-value of less than 0.05 was con-
sidered statistically significant.
3. RESULTS
A total of 30 women were enrolled, 15 in the study (rLH)
group and 15 in the control group. Two women from the
study group were removed from the study due to tech ni-
cal errors in taking their medication. There were no dif-
ferences between the two groups regarding the number
of previous IVF cycles and serum basal FSH and LH
levels (Table 1). Although women in the study group
were slightly older that those in the control group (32.5
± 2.3 years vs. 29.3 ± 3.6 years of age, respectively; p =
0.03), this difference does not seem to have any clinical
relevance (Table 1). The sperm parameters of women’s
partners were also similar. Total serum E2 concentration
and the serum E2 concentration per retrieved oocyte on
the day of hCG administration was higher but not sig-
nificant in the rLH group compared to the control group
(1461.0 ± 754 pg/ml vs.1088 ± 601 pg/ml; and 250.7 ±
156.0 pg/ml vs. 161.2 ± 83.4 pg/ml;, respectively) (Ta-
ble 2). The serum progesterone levels were not signifi-
cantly different between groups; 0.9 ± 0.6 pg/ml in rLH
group and 0 .7 ± 0.4 pg /ml in the con trol grou p. The total
amount of rFSH used during ovarian stimulation, as well
as the number of ovarian stimulation days was similar in
both groups. In all other cycle characteristics, no sig-
nificant differences were found between the two groups.
Serum LH levels in both groups decreased signifi-
cantly from basal levels on day three of the menstrual
cycle to the levels on the day of hCG injection. In study
group patients, the mean serum LH level decreased from
5.2 ± 2.2 IU/ml on cycle day three to 2.9 ± 1.6 IU/ml on
the first day of GnRH antagonist administration and to
2.4 ± 1.5 IU/ml on the day of hCG administration. Pa-
tients from the control group also showed reduced serum
LH levels from 4.9 ± 1.9 IU/ml on day three to 2.2 ± 1.2
IU/ml on day of GnRH antagonist initiation and 1.4 ±
1.0 IU/ml on day of hCG. However, the decrease in se-
rum LH was not significantly different between the two
groups (Figure 1).
Table 1. Patient characteristics.
Study
group Control group P value
Age (years) 32.5 ± 2.3 29.3 ± 3.6 0.03
Primary infertility71.4% 69.2% NS
Previous IV F cycles
(mean±sd) 3.0 ± 2.4 2.9 ± 2.4 NS
Basal FSH IU/L 7.4 ± 1.8 6.5 ± 1.9 NS
Basal LH IU/L 5.2 ± 2.2 4.9 ± 1.9 NS
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A. W iser et al. / Open Journal of Obstetrics and Gynecology 1 (2011) 31-35 33
Table 2. Cycle characteristics during the IVF treatment.
Study group
(N = 15) Control group (N = 13) P value
Peak E2 level on hCG (pg/ml) 1461 ± 754 1088 ± 601 NS
E2 concentration, per retrieved oocyte (ng/ml) 250.7 ± 156.0 161.2 ± 83.4 0.08
Serum progesterone on hCG (ng/ml) 0.9 ± 0.6 0.7 ± 0.4 NS
Endometrial thickness on hCG (mm) 9.2 ± 1.7 10.0 ± 2.0 NS
rFSH (total IU) 1800 ± 784 1960 ± 445 NS
Days of stimulation 9.3 ± 2.4 9.8 ± 1.8 NS
Mean number of retrieved oocytes 6.3 ± 1.6 8.3 ± 6.1 NS
Rate of ICSI 48% 36.8% NS
Fertilization rate 62.0% 52.4% NS
Embryos available for transfer (36/43) 84% (40/68) 59% 0.006
Mean number of embryos transferred (ET) 2.1 ± 0.6 1.9 ± 0.8 NS
Percentage of cr yopreserved embryos (14/43) 33% (11/68) 16% 0.005
Figure 1. LH level during ovarian stimulation.
The mean number of oocytes retrieved and fertiliza-
tion rates were not significantly different between the
rLH and control groups (6.3 ± 1.6 vs. 8.3 ± 6.1 and
62.0% vs. 54.4%, respectively). The number of embryo s
transferred (ET) was similar in both groups (mean 2.1 ±
0.6 in the rLH group compared to 1.9 ± 0.8 in the Con-
trol group). However, the percentage of grade 3-4
top-quality embryos available for transfer, out of all fer-
tilized oocytes was significantly higher, (36/43, 84%) in
the rLH group compared to the control group (40/68,
59%, p = 0.006). Moreover, the percentage of remaining
embryos those were suitable for Cryopreservation after
embryo transfer, was significantly higher in the rLH
group (14/43, 33%) compared to the control group
(11/68, 16%, p = 0.05; Table 2).
Hormonal concentrations of FSH and LH in the fol-
licular fluid were similar in both groups (Table 3). Ho w-
ever, there was a trend towards higher progesterone and
E2 concentrations in the follicular fluid in the study
group in compared to the control group (16.5 ± 2.5
µg/ml vs. 11.4 ± 3.6 µg/ml; P = 0.07 and 687 ± 112 pg/m
vs. and 471 ± 65 pg/m l , P = 0. 08 ) respectively.
Table 3. Hormone concentrations in follicular fluid.
Study groupControl group valueP
FSH (IU/ml)3.2 ± 0.7 3.2 ± 0.8 NS
LH (IU/ml) 4.5 ± 0.7 4.9 ± 0.5 NS
Progesterone
(nmol/ml) 16.5 ± 2.5 11.4 ± 3.6 P = 0.07
Estradiol level
(pg/ml) 687 ± 112 471 ± 65 P = 0.08
OJOG

A. W iser et al. / Open Journal of Obstetrics and Gynecology 1 (2011) 31-35 34
4. DISCUSSIONS
This study demonstrates that rLH supplementation dur-
ing GnRH antagonist cycles may improve embryo qual-
ity.
It is well established that FSH and LH play separate
but complementary roles in folliculogenesis. The “two
cell, two gonadotropin theory” suggests that the interac-
tion between FSH and LH is crucial for appropriate fol-
liculogenesis and oocyte maturation. Throughout most
of the follicle’s development, LH responsiveness is re-
stricted to the thecal cells that are differentiated in the
follicular pre-antral stage. During folliculogenesis, an-
drogens are produced in the thecal cells of antral folli-
cles in response to LH stimulation [8]. Androgens have
been shown to stimulate early follicular development
and reduce the incidence of apoptosis. High androgen
concentrations (or high androgen/estrogen ratios) have
been observed in lower quality oocytes [9].
Studies comparing rFSH to urinary gonadotropins
(LH and FSH) in down-regulated cycles with GnRH
agonist protocol [10] and mainly in GnRH agonist long
protocol [11] have been performed. Nevertheless, very
few studies reported on rLH addition in GnRH antago-
nist cycles [6,7]. One study [7] found no evident benefit
to rLH supplementatio n during GnRH antagonist cycles.
However, in that study one injection of long acting 3mg
cetrorelix was used. On the other hand, Acevedo et al. [6]
described that rLH supplementation improved the num-
ber of top quality embryos in recipients whose embryos
originated from GnRH antagonist-treated donors. This
unique model of donor cycles eliminated the endometrial
tissue factor and isolated the effect of LH supplementa-
tion on the ovary alone. This finding of a higher rate of
top quality embryos in patients who were treated with
rLH is also supported by our results. The MERIT study
[5] also found that supplementing ovarian stimulation
with LH has a beneficial effect on embryo quality;
however, in that study a long GnRH agonist protocol
was used. Moreover, HMG was used and not recombi-
nant LH and in contrast to our study, it was initiated on
the first day of ovarian stimulation.
The mechanism of how LH activity mediates im-
provements of oocyte and embryo quality parameters in
IVF is not fully understood. It is speculated that a set of
cumulus genes may determine oocyte maturation, fer-
tilization potential, and embryo quality [12]. Data from
sibling human oocytes suggest that embryo quality im-
proves when oocytes are allowed to intercalate with cu-
mulus cells, indicating an improvement o f cytoplasmatic
maturation [13]. Data fr om a gene expression study pro-
vided some molecular evidence for a mediation of cu-
mulus cells in embryonic development [14]. It has been
proposed that LH activity might influence the cumulus
cells surrounding the oocyte [15], affecting the oocyte-
cumulus interaction, the cytoplasmatic maturation of the
oocyte, and the quality of its develop ment. Cumulus cell
gene expression may provide a direct assessment of fer-
tility potential and a measure of embryo quality.
Another mechanism for LH activity is related to an-
drogen production. Androgens are produced by the theca
cells in response to LH. One study described that eleva-
tion of intrafollicular androgen concentration in early
follicular phase, resulted in a modest increase in the
number of good quality embryos [16]. This mechanism
could be, in part, the explanation of our results that
demonstrated an increase in the quality of embryos
among the LH gr oup.
Previous studies have reported on serum hormonal
concentration. Cédrin-Cédrin-Durnerin et al. [7] de-
scribed higher serum peak E2 level in patients treated in
with rLH. Bosch et al. [17] found that purified hMG,
resulted in higher serum E2 levels but lower progester-
one levels compared to the recombinant FSH group. In
our patients, the serum E2 level showed a trend toward
being higher in the rLH group: no difference was found
in progesterone levels between the groups. The differ-
ence in the results regard ing progesteron e levels between
our study and that of Bosch, et al. [17] could be due to
the different protocols used. Bosch et al. started with
HMG from the first day of ovarian stimulation but in our
study, rLH was added the day GnRH was admini st ered.
Our study offered the opportunity to correlate follicu-
lar fluid hormone concentrations and embryo quality. We
have shown that in antagonist cycles, LH supplementa-
tion led to increased follicular fluid levels in both E2 and
progesterone. This is not in agreement with the findings
of Smitz et al., [18] who reported an increased follicular
level of E2 but decreased level of progesterone in hMG
compared to rFSH. However, in that stud y a long GnRH
agonist protocol was used and that may explain the dif-
ferent results. It has been shown that E2 plays an impor-
tant role in ovarian cell differentiation [19] and oocyte
maturation [19]. Adding recombinant LH in a GnRH-
antagonist cycle provides sufficient substance products
to sustain the synthesis of E2, which is necessary for
oocyte maturation and differentiation [17].
Serum progesterone level was also found to be a
prognostic factor in IVF cycles. Niu et al. [20] reported
that women with a high serum progesterone concentra-
tion on the day of ovum pick up had a greater number of
viable embryos. In our study, follicular fluid levels of
both progesterone and E2 were higher in the rLH group.
This may partially explain the higher rates of top-quality
embryos derived from the LH group.
In conclusion, our study suggests that rLH supple-
mentation on the day of GnRH-antagonist initiation can
yield a higher number of top-quality embryos. Addi-
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A. W iser et al. / Open Journal of Obstetrics and Gynecology 1 (2011) 31-35 35
tional, larger studies are needed to determine the effect
on pregnancy and delivery rates.
OJOG
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