Recombinant human luteinizing hormone supplementation may improve embryo quality in in vitro fertilization/intracytoplasmic sperm injection cycles with gonadotropin-releasing hormone antagonist protocol

doi:10.4236/ojog.2011.12007

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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.

Email: [email protected]

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

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%

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

A. W iser et al. / Open Journal of Obstetrics and Gynecology 1 (2011) 31-35

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%

Fertilization rate 62.0% 52.4%

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

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-

A. W iser et al. / Open Journal of Obstetrics and Gynecology 1 (2011) 31-35

tional, larger studies are needed to determine the effect

on pregnancy and delivery rates.

OJOG

REFERENCES

[1] Weghofer, A., Munné, S., Brannath, W., Chen, S., Tom-

kin, G., Cekleniak, N., et al. (2008) The impact of

LH-containing gonadotropins on diploidy rates in preim-

plantation embryos: Long protocol stimulation. Human

Reproduction, 23, 499-503. doi:10.1093/humrep/dem412

[2] Manna, C., Rahman, A., Sbracia, M., Pappalardo, S.,

Mohamed, E.I., Linder, R., et al. (2005) Serum luteiniz-

ing hormone, follicle-stimulating hormone and oestradiol

pattern in women undergoing pituitary suppression with

different gonadotrophin-releasing hormone analogue pro-

tocols for assisted reproduction. Gynecological Endocri-

nology, 20, 188-194. doi:10.1080/09513590400027141

[3] Albano, C., Smitz, J. , Camus, M., Riethmüller-Winzen H,

Van Steirteghem A and Devroey P. (1997) Comparison of

different doses of gonadotropin-releasing hormone an-

tagonist Cetrorelix during controlled ovarian hyper-

stimulation. Fertility and Sterility, 6, 917-922.

doi:10.1016/S0015-0282(97)81407-0

[4] Kolibianakis, E.M., Venetis, C.A. and Tarlatzis, B.C.

(2009) Role of the endocrine profile for the achievement

of pregnancy with IVF. Reproductive BioMedicine On-

line, 18, 37-43. doi:10.1016/S1472-6483(10)60447-6

[5] Ziebe, S., Lundin, K., Janssens, R., Helmgaard, L. and

Arce, J.C. (2007) MERIT (Menotrophin vs Recombinant

FSH in vitro Fertilisation Trial) Group. Influence of ova-

rian stimulation with HP-hMG or recombinant FSH on

embryo quality parameters in patients undergoing IVF.

Human Reproduction, 22, 2404-2413.

doi:10.1093/humrep/dem221

[6] Acevedo, B., Sanchez, M., Gomez, J.L., Cuadros, J.,

Ricciarelli, E. and Hernández, E.R. (2004) Luteinizing

hormone supplementation increases pregnancy rates in

gonadotropin-releasing hormone antagonist donor cycles.

Fertility and Sterility, 82, 343-347.

doi:10.1016/j.fertnstert.2004.03.020

[7] Cédrin-Durnerin, I., Grange-Dujardin, D., Laffy, A.,

Parneix, I., Massin, N., Galey, J., et al. (2004) Recombi-

nant human LH supplementation during GnRH antago-

nist administration in IVF/ICSI cycles: A prospective

randomized study. Human Repr oduction, 19, 1979-1984.

doi:10.1093/humrep/deh369

[8] Durnerin, C.I., Erb, K., Fleming, R., Hillier, H., Hillier,

S.G., Howles, C.M., et al. (2008) Luveris Pretreatment

Group. Effects of recombinant LH treatment on follicu-

logenesis and responsiveness to FSH stimulation. Human

Reproduction, 23, 421-426. doi:10.1093/humrep/dem388

[9] McNatty, K.P., Smith, D.M., Makris, A., Osathanondh, R.

and Ryan, K.J. (1979) The microenvironment of the hu-

man antral follicle: Interrelationships among the steroid

levels in antral fluid, the population of granulosa cells,

and the status of the oocyte in vivo and in vitro. The

Journal of Clinical Endocrinology & Metabolism, 49,

851-860. doi:10.1210/jcem-49-6-851

[10] Hompes, P.G., Broekmans, F.J., Hoozemans, D.A. and

Schats. R. (2008) FIRM group. Effectiveness of highly

purified human menopausal gonadotropin vs. recombi-

nant follicle-stimulating hormone in first-cycle in vitro

fertilization-intracytoplasmic sperm injection patients.

Fertility and Sterility, 89, 1685-1693.

doi:10.1016/j.fertnstert.2007.05.039

[11] Andersen, A.N., Devroey, P. and Arce, J.C. (2006) Clini-

cal outcome following stimulation with highly purified

hMG or recombinant FSH in patients undergoing IVF: A

randomized assessor-blind controlled trial. Human Re-

production, 21, 3217-3227.

doi:10.1093/humrep/del284

[12] McKenzie, L.J., Panga s, S.A., Carson, S.A., Kovanci, E.,

Cisneros, P., Buster, J.E., Amato, P. and Matzuk, M.M.

(2004) Human cumulus granulosa cell gene expression:

A predictor of fertilization and embryo selection in

women undergoing IVF. Human Reproduction, 19, 2869-

2874. doi:10.1093/humrep/deh535

[13] Hassan, H.A. (2001) Cumulus cell contribution to cyto-

plasmic maturation and oocyte developmental compe-

tence in vitro. Journal of Assisted Reproduction and Ge-

netics, 18, 539-543.

[14] Assou, S., Anahory, T., Pantesco, V., Le Carrour, T.,

Pellestor, F., Klein, B., et al. (2006) The human cumu-

lus—Oocyte complex gene-expression profile. Human

Reproduction, 21, 1705-1719.

doi:10.1093/humrep/del065

[15] Platteau , P., Smitz , J., Albano, C., Sørensen, P., Arce, J.C.

and Devroey, P. (2004) Exogenous luteinizing hormone

activity may influence the treatment outcome in in vitro

fertilization but not in intracytoplasmic sperm injection

cycles. Fertility and Sterility, 81, 1401-1404.

doi:10.1016/j.fertnstert.2003.09.077

[16] Lossl, K., Andersen, A.N., Loft, A., Freiesleben, N.L.,

Bangsbøll, S. and Andersen, C.Y. (2006) Androgen

priming using aromatase inhibitor and hCG during early-

follicular-phase GnRH antagonist down-regulation in

modified antagonist protocols. Human Reproduction, 21,

2593-2600. doi:10.1093/humrep/del221

[17] Bosch, E., Vidal, C., Labarta, E., Simon, C., Remohi, J.

and Pellicer, A. (2008) Highly purified hMG versus re-

combinant FSH in ovarian hyperstimulation with GnRH

antagonists—A randomized study. Human Reproduction,

23, 2346-2351.

doi:10.1093/humrep/den220

[18] MERIT* Group. (2007) Endocrine profile in serum and

follicular fluid differs after ovarian stimulation with

HP-hMG or recombinant FSH in IVF patients. Human

Reproduction, 22, 676-687.

[19] Couse, J.F., Hewitt, S.C., Bunch, D.O., Sar, M., Walker,

V.R., Davis, B.J., et al. (1999) Postnatal sex reversal of

the ovaries in mice lacking estrogen receptors alpha and

beta. Science, 17, 2328-2331.

[20] Niu, Z. , Feng, Y., Zhang, A., Sun, Y. and Zhang, H. (2008)

Progesterone levels on oocyte retrieval day can predict

the quantity of viable embryos but not pregnancy out-

come of intracytoplasmic sperm injection. Gynecological

Endocrinology, 24, 452-458.

doi:10.1080/09513590802196247