Assisted Reproduction-In Vitro Fertilization ... - Endocrine Reviews

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Endocrine Reviews 20(3): 249–252
Copyright © 1999 by The Endocrine Society
Printed in U.S.A.
Assisted Reproduction-In Vitro Fertilization Success Is
Improved by Ovarian Stimulation with Exogenous
Gonadotropins and Pituitary Suppression with
Gonadotropin-Releasing Hormone Analogues*
ROBERT L. BARBIERI AND MARK D. HORNSTEIN
Department of Obstetrics, Gynecology and Reproductive Biology, Brigham and Women’s Hospital,
Harvard Medical School, Boston, Massachusetts 02115-6110
I. Introduction
II. Ovarian Stimulation with Exogenous Gonadotropin Improves
In Vitro Fertilization and Embryo Transfer (IVF-
ET) Success
III. Pituitary Suppression Improves IVF-ET Success: GnRH
Agonist Analogues
IV. Pituitary Suppression Improves IVF-ET Success: GnRH
Antagonist Analogues
I. Introduction
THE ESSENCE of mammalian reproduction is the fusion
of a sperm and oocyte to form a conceptus. In this
miraculous process, the single-cell conceptus contains all the
biological information necessary to produce a new life. In
nature, the important processes in mammalian reproduction
occur within the bodies of the female and male partners. The
essence of assisted reproduction is that a third party, the
reproductive endocrinologist-biologist, directly manipulates
the sperm and/or oocyte to enhance the probability of
achieving a pregnancy. Although assisted reproduction appears
to be a new field, it was being practiced before the
discovery of insulin. In 1890, Heape (1) reported the successful
transfer of embryos from a donor rabbit to a recipient,
resulting in the first birth by a surrogate gestational carrier.
In 1944, Rock and Menkin (2) reported the successful fertilization
of human oocytes in vitro and their development to
the two- and three-cell stage. In 1959, Chang (3) successfully
fertilized a rabbit oocyte in vitro. In humans, the successful
capacitation of sperm in vitro and the successful fertilization
of human oocytes matured in vitro (4) were followed by the
discovery that preovulatory oocytes were relatively easy to
fertilize in vitro (5). These discoveries culminated in 1978 with
the birth of a baby girl resulting from the in vitro fertilization
of a single preovulatory oocyte obtained from a natural menstrual
cycle (6).
Assisted reproduction has evolved rapidly over the past
two decades with significant progress in our understanding
Address reprint requests to: Robert Barbieri, M.D., Department of
Obstetrics and Gynecology, ASB1–3, Brigham and Women’s Hospital, 75
Francis Street, Boston, Massachusetts 02115 USA. e-mail: RLBarbieri@
BICS.BWH.Harvard.edu
* Supported in part by NIH Grant U54HD-29164.
249
and techniques in three major areas: 1) the endocrinology of
the assisted reproduction process; 2) the biology of gamete
function and the technology of gamete micromanipulation;
and 3) the genetics of reproduction. This minireview will
focus on advances in the endocrinology of the assisted reproductive
process with an emphasis on data from well
designed (randomized) clinical trials. The specific focus will
be on data that demonstrate that in humans, given our current
technology, successful in vitro fertilization and embryo
transfer (IVF-ET) requires both stimulation of the ovary and
suppression of the pituitary.
II. Ovarian Stimulation with Exogenous
Gonadotropins Improves In Vitro Fertilization and
Embryo Transfer (IVF-ET) Success
A characteristic feature of IVF-ET is that a very clear and
easily measurable endpoint (birth of a normal newborn) is
the goal of the treatment. There are few endocrine treatments
with such a clearly relevant and precisely measured endpoint.
Many factors influence the success of IVF-ET including
the age of the female partner (7), early follicular phase FSH
concentration (8), evidence of good ovarian reserve (9), and
the number of oocytes retrieved. An important determinant
of IVF-ET success is the ovarian stimulation regimen employed.
Although the first birth from IVF-ET used a single oocyte
obtained from a natural cycle, most studies demonstrate that
the pregnancy rate is very low when natural cycles are used
for IVF. In one randomized study, IVF success was compared
using a natural cycle vs. a clomiphene citrate-stimulated cycle.
In the group of women randomized to treatment in a
natural cycle, 0.3 oocytes were retrieved per cycle and no
pregnancies occurred. In the group randomized to the clomiphene-stimulated
cycles, 1.8 oocytes were retrieved per
cycle and there was a 6% pregnancy rate (10). Even with
refinements in natural cycle IVF-ET, the average pregnancy
rate per cycle initiated tends to be in the 3–10% range (11–13).
This low success rate makes natural cycle IVF-ET unsuitable
for clinical use, except in unusual clinical circumstances such
as reluctance of the female partner of the infertile couple to
use agents that stimulate ovarian follicular growth.
Clomiphene citrate as a single agent has been evaluated for

250 BARBIERI AND HORNSTEIN Vol. 20, No. 3
ovarian stimulation in IVF-ET in very few clinical trials. As
noted above, use of clomiphene citrate as a single agent for
ovarian stimulation resulted in a pregnancy rate per cycle of
6% (10). In other small clinical trials the use of clomiphene for
ovarian stimulation in IVF-ET typically results in pregnancy
rates per cycle in the range of 6% (14). These pregnancy rates
are far lower than rates achieved by stimulation protocols
that use exogenous gonadotropins for ovarian stimulation.
One explanation for these observations is that in IVF-ET,
clomiphene stimulation does not produce enough mature
oocytes to maximize the chance of pregnancy. Clomiphene
has also been used in conjunction with pulsatile administration
of the native decapeptide GnRH at doses of 14 �g
every 90 min. The use of clomiphene followed by pulsatile
GnRH resulted in the stimulation of five to seven large follicles
and can be associated with pregnancy (15). Use of
pulsatile GnRH requires a programmable infusion pump
and chronic parenteral access. These factors have limited its
use in IVF-ET.
Most IVF programs now utilize exogenous gonadotropins
for ovarian stimulation. Gonadotropin preparations that are
commonly used include human menopausal gonadotropins
(Pergonal, Serono, Randolph, MA; Humegon, Organon,
West Orange, NJ; Repronex, Ferring, Tarrytown, NY), highly
purified urinary FSH (Fertinex, Serono) and recombinant
FSH (Gonal-F-Serono, Follistim-Organon). There are few
randomized clinical trials that evaluate the efficacy of exogenous
gonadotropins against clomiphene alone or natural
cycles alone in IVF-ET. However, in IVF-ET, ovarian stimulation
regimens that include human gonadotropins are routinely
associated with pregnancy rates per cycle in the range
of 20% to 50%. These pregnancy rates are far greater than
those observed when clomiphene is used alone or natural
cycles are used for IVF-ET.
III. Pituitary Suppression Improves IVF-ET Success:
GnRH Agonist Analogues
Many randomized clinical trials demonstrate that in IVF-
ET, the combination of exogenous gonadotropin plus a
GnRH agonist for suppression of pituitary LH and FSH secretion
is associated with higher pregnancy rates than the use
of gonadotropins without a GnRH agonist. Most GnRH agonist
analogues differ from the native decapeptide GnRH in
amino acid positions 6 and 10 and are resistant to degradation
by endopeptidases, thus giving them long half-lives. In
FIG. 1. Schematic representation of
the long GnRH agonist analogue protocol
with step-down gonadotropin stimulation.
[Reproduced with permission
from O. K. Davis and Z. Rosenwaks: In
vitro fertilization. In: Keye WR, Chang
RJ, Rebar RW, Soules MR (eds) Infertility:
Evaluation and Treatment. WB
Saunders, Philadelphia, 1995, p 763.]
addition, the GnRH agonist analogues have high affinity for
the receptor and long receptor occupancy (16). The initial
administration of GnRH agonist analogues is associated with
an increase in LH and FSH secretion (agonist phase). Prolonged
administration causes down-regulation and partial
desensitization of the pituitary GnRH receptor, resulting in
the suppression of LH and FSH secretion. The addition of
GnRH agonist analogues to regimens of gonadotropin stimulation
for IVF-ET appears to be associated with an increase
in the number of oocytes retrieved, the number of embryos
transferred, and the clinical pregnancy rate.
For example, one study of IVF-ET demonstrated that treatment
with a GnRH agonist (buserelin) plus hMG resulted in
more oocytes retrieved (9.3 vs. 6.2), more embryos (4.3 vs.
2.8), and a higher clinical pregnancy rate (20% vs. 14%) than
stimulation with hMG in the absence of buserelin (17). In
another clinical trial that used the same medications, treatment
with a GnRH agonist plus hMG resulted in a higher
pregnancy rate than treatment with hMG without a GnRH
agonist (36% vs. 18%) (18). In a meta-analysis of 15 clinical
trials evaluating the impact of a GnRH agonist on IVF-ET,
Hughes and colleagues (19) reported that the available data
support the routine use of pituitary suppression in assisted
reproduction including IVF-ET and gamete intrafallopian
tube transfer (GIFT). The Hughes meta-analysis demonstrated
that the use of GnRH agonists in the ovarian stimulation
protocols reduced cycle cancellation rate by 67% and
increased IVF-ET clinical pregnancy rate by approximately
70%. Figure 1 is a schematic representation of a common
protocol combining GnRH agonist suppression of pituitary
gonadotropin secretion with exogenous gonadotropin administration
to stimulate ovarian follicular growth.
The biological mechanisms that subserve these clinical
findings are not fully characterized. However, with gonadotropin
stimulation of ovarian multifollicular development,
it is common to observe premature surges of LH. Surges of
LH that occur before full follicle maturity may cause premature
luteinization of the granulosa cells, which may be
detected in some cases by an increase in circulating progesterone
before full follicle maturation. In addition, a premature
surge of LH may disrupt oocyte maturation (20, 21). The
suppression of pituitary gonadotropin secretion with a
GnRH analogue permits longer ovarian stimulation, which
results in the development of a greater number of large
mature follicles. In turn, this permits the retrieval of a greater
number of competent oocytes, which increases the number

June, 1999 IMPROVEMENTS IN IVF-ET 251
TABLE 1. Dose effects of a GnRH antagonist on pregnancy success in IVF-ET
Group
1 2 3 4 5 6
Dose of ganirelix (mg) 0.0625 0.125 0.25 0.50 1.0 2.0
Number of women 30 65 68 69 64 26
FSH (IU/liter) 9.1 9.0 9.1 10.2 9.8 8.8
LH (IU/liter) 3.6 2.5 1.7 1.0 0.6 0.4
Androstenedione (ng/ml) 2.6 2.6 2.4 2.2 2.0 1.5
Estradiol (pg/ml) 1475 1130 1160 823 703 430
Pregnancy rate per cycle (%) 23.3 23.1 33.8 10.1 14.1 0
Women undergoing IVF received recombinant FSH (Puregon) starting on cycle day 2 and continuing daily until three follicles with a mean
diameter �17 mm, as determined by ultrasound, were achieved when hCG was given to initiate the ovulatory process. The women were
randomized to receive the GnRH antagonist, ganirelix, on a daily basis starting on cycle day 7 at one of six doses (0.0625 mg, 0.125 mg, 0.25
mg, 0.5 mg, 1.0 mg, 2.0 mg). Serum FSH, LH, androstenedione, and estradiol concentration were measured on the day of hCG administration.
Large doses of ganirelix markedly suppressed serum LH, androstenedione, and estradiol concentrations. Pregnancy rate per cycle was maximal
with a dose of ganirelix of 0.25 mg. [Derived from Ref. 27.]
of healthy embryos created and thereby improves the pregnancy
rate in IVF.
IV. Pituitary Suppression Improves IVF-ET Success:
GnRH Antagonist Analogues
A major problem with the GnRH agonist analogues is that
LH secretion is stimulated at the initiation of treatment. In
some women, prolonged daily use of a GnRH agonist may
cause a small increase in LH secretion directly after the daily
administration of the GnRH agonist. In turn, residual pituitary
LH secretion stimulates ovarian androgen production,
which may have detrimental effects on follicular development
and endometrial function (22). The GnRH antagonists
offer the possibility of acutely suppressing LH secretion
without an initial increase in LH secretion (23, 24). The GnRH
antagonists have been used either as small daily doses (cetrorelix,
0.25 mg daily sc injection) during the early or midfollicular
phases of the stimulation cycle (25) or as a single
dose (cetrorelix, 3 mg sc) on approximately cycle day 8 (26).
Both regimens block the occurrence of spontaneous LH
surges.
The GnRH antagonists suppress LH secretion in a dosedependent
manner. At small doses, the suppression of LH is
minimal. At large doses, near-complete suppression of LH
can be achieved. In one study the impact of six doses of the
GnRH antagonist ganirelix on LH secretion and IVF outcomes
was studied (27). Ganirelix produced a dose-dependent
suppression of LH (Table 1). Ganirelix also produced a
dose-dependent suppression of both androstenedione and
estradiol. The larger doses of ganirelix were associated with
a markedly reduced pregnancy rate. These data support the
importance of both FSH and LH in the development of the
ovarian follicle. Ovarian estradiol production requires the
coordinated action of LH on the ovarian theca to stimulate
the production of androstenedione and FSH on the granulosa
cells to stimulate the aromatization of the androstenedione to
estrogen. Large doses of GnRH antagonists can nearly ablate
LH secretion, resulting in a reduction in follicular androstenedione
and estradiol production. When LH secretion is
completely blocked, pregnancy rates appear to be reduced.
These findings support the hypothesis that both high LH
levels (premature LH surge) and very low LH levels can be
associated with low pregnancy rates in IVF-ET. A major
advantage of the GnRH antagonists is that they can acutely
reduce LH secretion. This effect is not possible with the
GnRH agonist analogues. The ultimate role of the GnRH
antagonists in IVF-ET will require large prospective studies
to directly compare the efficacy of the GnRH agonists vs.
GnRH antagonists in IVF-ET.
A major goal of reproductive endocrinologists is to assist
infertile couples to build their families. As the cost of IVF-ET
decreases and the success of this procedure increases, the
importance of IVF-ET in the treatment of infertility will continue
to increase. The data reviewed in this article suggest
that exogenous gonadotropins and GnRH analogues are the
key hormones required to maximize IVF-ET success.
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International Workshop on Estrogens and Male Reproduction (EMR)
September 23rd and 24th, 1999
Caporizzuto, Calabria, Italy
This event will involve international experts discussing topics dealing with:
Aromatase gene expression in male gonad and extragonadal tissue
Estrogen production, estrogen receptors in male gonad and extragonadal tissue
Estrogen and male gonad development
Estrogen, xenoestrogens and male fertility
Estrogens and growth in males
Estrogens and bone in males
Deadline for abstract presentation: June 15, 1999
Deadline for early registration at a reduced fee: June 30, 1999
For more information, contact: Prex s.r.1., Viale Monza, 137, 20125 Milan, Italy. Telephone: �39-02-28311821.
Fax: �39-02-28311840. E-mail: congressi@prex.it Web site: www.prex.it/emr.htm