Gonadotropin-releasing hormone agonist/antagonist

conversion with estrogen priming in low responders

with prior in vitro fertilization failure
Jeffrey D. Fisch, M.D.,a Levent Keskintepe, Ph.D.,a and Geoffrey Sher, M.D.a,b
a
Sher Institute for Reproductive Medicine, Las Vegas; b Department of Obstetrics and Gynecology,
University of Nevada School of Medicine, Reno, Nevada

Objective: To evaluate gonadotropin-releasing hormone (GnRH) agonist/antagonist conversion with estrogen

priming (AACEP) in low responders with prior IVF failure.
Design: Descriptive.
Setting: Private practice.
Patient(s): Women aged %42 with prior IVF attempts in which all embryos were <7 cells or >20% fragmentation
on day 3 (n ¼ 137; <38: n ¼ 63; 38-42: n ¼ 74). In addition to unexplained poor response to stimulation (n ¼ 52),
diagnoses included elevated follicle-stimulating hormone (FSH >9.0 mIU/mL; n ¼ 40), advanced age (>41 years;
n ¼ 26), endometriosis (III–IV; n ¼ 12), and decreased ovarian reserve (AFC <5; n ¼ 7).
Intervention(s): Patients received sequential GnRH agonist, low-dose GnRH antagonist, and estradiol valerate
followed by recombinant FSH, 600 IU/day (n ¼ 72) or 750 IU/day (n ¼ 65).
Main Outcome Measure(s): Pregnancy, ongoing gestation, implantation rates.
Result(s): Although women aged <38 years and those on 600 IU/day produced more mature eggs and fertilized
embryos than women aged 38 to 42 years, there were no differences in peak estradiol, endometrial lining, or
embryos transferred. Outcomes were similar for all patients regardless of age or FSH dosage. Ongoing gestation
rates were 27% (37 out of 137) for all patients, 25% (16 out of 63) for age <38 years, and 28% (21 out of 74) for
ages 38 to 42 years.
Conclusion(s): Women aged %42 years who are candidates for oocyte donation may still achieve pregnancy using
their own eggs with the AACEP protocol. (Fertil Steril 2008;89:342–7. 2008 by American Society for Repro-
ductive Medicine.)
Key Words: GnRH agonist, GnRH antagonist, estrogen priming, IVF failure

It is well established that women are born with all the eggs
they will ever have. That these eggs are arrested in prophase
I of meiosis and must ultimately reduce their chromosome
number by half before fertilization can occur is not generally
as well appreciated (1). As eggs age, nondisjunctional events
at the time of ovulation occur more frequently (2–4). This
risk becomes so significant that women over the age of 35
years are routinely offered screening to detect fetal aneu-
ploidy. Nondisjunctional events at the first meiotic division
are the most common cause of first trimester miscarriage
(3) as well as the most common cause of failure of assisted
reproduction techniques (ART) (4).
Embryos that are morphologically abnormal are almost
always aneuploid, although many embryos appearing normal
on day 3 (>7 cells, <20% fragmentation) or day 5 are still
genetically abnormal (5–6). Because most genetic errors oc-
cur at ovulation, manipulation of the hormonal environment
surrounding the oocyte before ovulation may increase the
chance of that oocyte completing meiosis normally. Primary
follicles are metabolically inactive, and follicular growth
Received June 6, 2006; revised and accepted March 2, 2007.
Reprint requests: Jeffrey D. Fisch, M.D., Sher Institute for Reproductive
Medicine, Las Vegas, 3121 S. Maryland Parkway, Suite 300, Las Vegas,
NV 89109 (FAX: 702-892-9666; E-mail: jfisch@sherinstitute.com).
occurs in waves. Because the outcome of meiosis is not deter-
mined until fertilization, if even some of these inactive
follicles can be presumed genetically normal, then women
with a history of aneuploid eggs could potentially still
produce genetically normal ones in a subsequent cycle.
The most prevalent approaches for treating poor-prognosis
patients at present are the short protocol, also known as the
microdose gonadotropin-releasing hormone (GnRH) agonist
‘‘flare’’ (7, 8), and the GnRH antagonist protocol (9–12).
Neither of these protocols has been especially effective in
improving ART outcomes in these patients (13–15). The
initial flare of follicle-stimulating hormone (FSH) in the short
protocol may be promoting follicular development, but the
luteinizing hormone (LH) flare that occurs with it may be si-
multaneously promoting apoptosis (14). In a similar fashion,
the standard antagonist protocol exposes the maturing
oocytes to the patient’s own endogenous androgen produc-
tion during the 6 to 7 days of stimulation before development
of a mature follicle (13). Even low levels of androgen could
be significant in patients who are overly sensitive.
We present a novel approach to treating patients with prior
poor response to ovarian stimulation based on the intrinsic
physiology and biochemistry of the ovary. Using a hypothesis
of androgen oversensitivity (as opposed to the androgen

342 Fertility and Sterility Vol. 89, No. 2, February 2008 0015-0282/08/$34.00
Copyright ª2008 American Society for Reproductive Medicine, Published by Elsevier Inc. doi:10.1016/j.fertnstert.2007.03.004
overproduction commonly seen in association with anovula-
tion), we developed a protocol promoting estrogen domi-
nance in the ovary. Using GnRH agonist/antagonist
conversion with estrogen priming (AACEP), we have been
able to achieve success in women failing the short protocol
or the standard GnRH antagonist approach.
Since 2002, we have used this AACEP protocol in our
poorest prognosis patients and have seen improvement in
egg quality compared with their prior response with other
standard protocols for poor responders. In 2004 we reported
results of a small randomized, controlled trial evaluating this
protocol (16). We achieved a 43% ongoing pregnancy rate in
the AACEP group, all of whom had been given a little chance
of ever conceiving with their own eggs. Since 2003 the AA-
CEP has been our standard approach for poor responders.
This work further describes our experience with the AACEP
protocol among a large cohort of women with poor prognosis
who were not ready to consider oocyte donation.
MATERIALS AND METHODS
Patients
Between January 1, 2002 and December 31, 2006, 137
women aged %42 years with prior IVF failure and poor
quality embryos were treated using their own eggs with an
AACEP protocol at our center by a single physician. All
patients in this analysis had only their first AACEP treatment
cycle included. Characteristics of the population are shown in
Table 1. Every patient was found to be a poor responder to
stimulation, with all transferred embryos in prior attempts
<7 cells or >20% fragmentation on day 3. In addition to
previous IVF failure, every patient in this cohort had an addi-
tional diagnosis of either advanced maternal age (R41 years;
n ¼ 26), decreased ovarian reserve (AFC <5; n ¼ 7), elevated
levels of FSH (>9.0 mIU/mL; n ¼ 40), endometriosis (stage
III–IV; n ¼ 12), or unexplained poor response to stimulation
(<6 mature follicles; n ¼ 52). Several patients, especially
those with advanced maternal age, had more than one diagno-
TABLE 1
Characteristics 137 low responding women aged %42 with prior IVF failure using agonist/antagonist
conversion with estrogen priming.
Characteristic
Patents treated
Mean prior IVF attempts (SD)
Diagnosis
Unexplained poor response
(<6 mature follicles)
Elevated FSH (R9.0 mIU/mL)
Advanced maternal age (R41 years)
Endometriosis (stage III–IV)
Decreased ovarian reserve (AFC <5)
Fisch. Agonist/antagonist conversion. Fertil Steril 2008.
Fertility and Sterility
sis. Those with unexplained failure had a presenting diagno-
sis of male factor infertility or tubal occlusion. Every patient
in this cohort had been counseled by her prior physician to
pursue oocyte donation and that further treatment with her
own eggs would be futile.
Ovarian Stimulation
All patients were pretreated with estrogen/progestin oral con-
traceptive pills for 1 to 3 weeks. The GnRH agonist (Lupron
0.5 mg/day; TAP Pharmaceuticals, Lake Forest, IL) was
started in a standard long protocol, overlapping the last 5 to
7 days of oral contraceptive. The GnRH agonist was stopped
with the onset of menses and replaced by low-dose (0.125
mg/day) GnRH antagonist on cycle day 2.
Estradiol valerate, 2 mg, was given intramuscularly every
3 days for two doses. Estrogen suppositories were used to
maintain the endometrium until at least one follicle measured
15 mm. Follicular development was then stimulated using re-
combinant FSH in initial doses of 600 or 750 IU/day, decreas-
ing to 225 IU/day after 5 days. After the first 2 days, hMG at
75 IU/day was substituted for the 75 IU/day of recombinant
FSH. Transvaginal ultrasound monitoring began after 7
days of stimulation. The average patient needed 13 days of
stimulation before hCG administration. No cases of over-
stimulation occurred. Ovulation was triggered using hCG
when at least one follicle was 18 mm and half of the remain-
der were 15 mm. Oocytes were recovered transvaginally
using ultrasound guidance 34.5 hours after trigger.
Embryo Culture
Metaphase II (MII) oocytes were fertilized using intracyto-
plasmic sperm injection (ICSI) 4 to 6 hours after retrieval
in all cases. Embryos were cultured individually in 50-mL
droplets of early cleavage medium (ECM) (Sage BioPharma,
Pasadena, CA) under oil at 37 C in a 5% CO2, 5% O2, 90%
N2 environment. All embryos were serially evaluated over
n Ongoing gestation n (%)
137 37 (27.0)
1.9 (1.3)
52 15 (28.9)
40 14 (35.0)
26 5 (19.2)
12 2 (16.7)
7 1 (14.3)
343
the 72 hours following ICSI, generating a graduated embryo
score (GES) (17). Embryos were transferred to blastocyst
medium (Irvine Scientific, Santa Ana, CA) 46 hours after
ICSI (18).
Embryo Transfer
The number of embryos transferred was ultimately decided
by the patient after informed consent and based on American
Society of Reproductive Medicine (ASRM) guidelines (19).
The cervical canal was cleansed of bacteria and mucous be-
fore transfer using warmed culture media. Embryos were
transferred on day 3 of culture under abdominal ultrasound-
guidance using a flexible catheter (Wallace, Smiths Medical
International, Hythe, Kent, United Kingdom). Serum beta-
human chorionic gonadotropin (b-hCG) levels were mea-
sured 11 and 13 days after egg retrieval. Two values above
5.0 IU were considered positive. Ongoing gestation was
defined as fetal heart activity at 12 weeks of gestation.
Statistical Analysis
A normally distributed population was estimated by the size
of the standard deviation (SD) relative to the mean and was
confirmed using the D’Agostino and Pearson omnibus nor-
mality test. Differences between groups were evaluated using
Student’s t-tests, and differences in rates and proportions
were evaluated using chi-squared tests. P<.05 was consid-
ered statistically significant.
Institutional Review
As conceived in 2002, this study was a randomized, con-
trolled trial of the AACEP protocol compared with our
then-standard agonist protocol with estrogen priming. This
study was approved and monitored by an institutional review
board. The preliminary data from this study were presented in
2004 (16). However, only the 20 patients in that group who
received an AACEP protocol are included in this analysis,
and only these patients signed a specific consent form
approved by the institutional review board. All of our patients
do routinely consent to collection of their de-identified data
for future research.
Beginning in January 2003, the AACEP was instituted as
the standard approach for low-responding patients at our cen-
ter who had had prior IVF failure due to poor embryo quality.
Every patient was counseled regarding the potential risks,
benefits, and alternatives to this protocol; however, because
it was no longer considered experimental, a specific institu-
tional review board approval was not sought prospectively.
We did seek an amendment to the previously approved
protocol allowing a records review.
RESULTS
In this cohort of 137 low responders with prior IVF failure
aged <38 years (n ¼ 63) and 38 to 42 years (n ¼ 74), there
were a total of 37 ongoing gestations (27%). The characteris-
tics of the population are shown in Table 1. The mean number
344 Fisch et al. Agonist/antagonist conversion
of prior IVF failures was 1.9. By diagnosis, ongoing gesta-
tions were achieved in 35% (14 out of 40) of women with
elevated FSH levels, 29% (15 out of 52) of women with unex-
plained poor response, 19% (5 out of 26) of women with
advanced maternal age, 17% (2 out of 12) of women with
severe endometriosis, and 14% (1 out of 7) of women
with decreased ovarian reserve.
When stratified by age, women <38 produced significantly
more metaphase II oocytes and fertilized embryos (6.9 and
5.4, respectively) than women 38 to 42 (5.3 and 4.1;
P<.05) (Table 2). However, there were no differences in
pregnancy, ongoing gestation, or implantation rates between
the two groups. Nor were there any differences in peak
estradiol, endometrial thickness, prior IVF attempts, or the
number of embryos transferred. The ongoing gestation rate
was 25% (16 out of 63) in women <38 years and 28% (21
out of 74) in women 38 to 42 years. Most women aged 38
to 42 years, (58%; 43 out of 74) received 750 IU/day of
recombinant FSH, but most women <38 years (65%; 41
out of 63) received 600 IU/day of recombinant FSH; this
difference was not statistically significant.
When stratified by FSH dose, patients receiving 600 IU/
day were younger (mean age: 36.1 vs. 37.6 years; P<.02)
and produced more mature oocytes (7.2 vs. 4.7; P<.002)
and fertilized embryos (5.7 vs. 3.5; P<.002) than women
receiving 750 IU/day (Table 3). There were no statistically
significant differences in the number of prior IVF attempts,
peak estradiol level, endometrial thickness, or the number
of embryos transferred. Pregnancy, ongoing gestation, and
implantation rates between the two groups were similar.
DISCUSSION
Developmental events in the follicle culminating in oocyte
reactivation are hormonally controlled by endocrine, para-
crine, and autocrine factors. In a natural ovulatory cycle,
the dominant follicle is the one most capable of creating an
estrogenic microenvironment surrounding the oocyte (20).
Ovarian theca cells can produce androgens, but only granu-
losa cells produce the P450arom necessary to catalyze these
androgens into estrogen (21). Aromatase activity is increased
by FSH and decreased by LH. Also, FSH and estradiol pro-
mote accumulation of granulosa cell–FSH receptors. Al-
though FSH alone promotes initial follicular development
(22), the final stages of maturation are optimized by LH
(23). Oocytes in an estrogenic microenvironment mature
and ovulate, whereas oocytes in an androgenic microenviron-
ment undergo apoptosis and atresia (24–26). Androgen dom-
inance in the early follicular phase promotes apoptosis
through a paracrine mechanism resulting in elevated tumor
necrosis factor alpha (TNF-a) expression (27, 28).
Male hormone exposure in the early proliferative phase is
detrimental to oocyte quality (29). In an androgenic environ-
ment, preantral follicles favor conversion to 5-a reduced an-
drogens rather than to estrogen (30). These androgens cannot
be further converted to estrogen, and they inhibit aromatase
Vol. 89, No. 2, February 2008
 TABLE 2
Characteristics and ART cycle outcomes of 137 fresh embryo transfers into low responding women
aged %42 with prior IVF failure using agonist/antagonist conversion with estrogen priming.
<38 38–42
Characteristic Mean (SD) Mean (SD)
Embryo transfer cycles (n) 63 74
FSH dosage: 600 IU/day (n) 41 31
750 IU/day (n) 22 43
Age (years) 33.6 (2.8) 39.6 (1.2)
Previous IVF failures 2.1 (1.2) 1.8 (1.3)
Peak estradiol (pg/mL) 1179 (667) 1105 (810)
Endometrial thickness (mm) 11.8 (2.1) 11.8 (2.0)
Metaphase II oocytes 6.9 (5.2)a 5.3 (3.7)
Fertilized embryos 5.4 (4.0)a 4.1 (2.9)
Embryos transferred 2.6 (1.1) 2.6 (1.1)
Pregnancy rate 44% (28/63) 51% (38/74)
Ongoing gestation rate 25% (16/63) 28% (21/74)
Implantation rate (sacs/ET) 18% (29/165) 18% (36/196)
a
P< .05 compared with age 38 to 42.
Fisch. Agonist/antagonist conversion. Fertil Steril 2008.

activity (31). Exposure to LH is also reported to down-regu-
late the formation of connexin gap junctions between granu-
losa cells (32) and close existing channels necessary for
coordinated follicular development (33).
Pretreatment with estrogen/progestin oral contraceptive
pill is important to establishing an estrogenic environ-
ment. The dual actions of progestin-mediated LH suppres-
sion and estrogen-mediated stimulation of sex hormone–
binding globulin result in decreased follicular androgen
levels. Oversuppression from the pill has been a potential
concern, but because its actions are central, direct ovarian
stimulation with exogenous FSH should overcome any
issues.
The GnRH agonist was retained in this protocol to take ad-
vantage of the FSH/LH ‘‘flare’’ effect occurring in the artifi-
cial luteal phase of oral contraceptive pretreatment, where it
may augment follicular recruitment. However, GnRH recep-
tors have been identified in the ovary, and, due to its relatively

TABLE 3
Characteristics and ART outcomes of 137 fresh embryo transfers stratified by FSH dosage into low
responding women aged %42 with prior IVF failure using agonist/antagonist conversion with estrogen
priming.
600 750
FSH dosage (IU/day) Mean (SD) Mean (SD)
Embryo transfer cycles (n) 72 65
Age (years) 36.1 (3.6)a 37.6 (3.5)
Previous IVF 1.8 (1.1) 2.1 (1.5)
Peak estradiol (pg/mL) 1197 (738) 1096 (753)
Endometrium (mm) 11.8 (2.1) 11.7 (1.9)
Mature oocytes 7.2 (4.7)b 4.7 (3.9)
Fertilized embryos 5.7 (3.8)b 3.5 (2.7)
Embryos transferred 2.8 (1.1) 2.5 (1.1)
Pregnancy rate 49% (35/72) 48% (31/65)
Ongoing gestation rate 29% (21/72) 25% (16/65)
Implantation rate (sacs/ET) 21% (41/200) 15% (24/161)
a
P< .02 compared with 750 IU/day.
b
P< .002 compared with 750 IU/day.
Fisch. Agonist/antagonist conversion. Fertil Steril 2008.

Fertility and Sterility 345

long half-life, GnRH agonist has been reported to suppress
follicular development during stimulation (34, 35). However,
removing pituitary suppression entirely may lead to prema-
ture luteinization from endogenous androgens, especially in
patients hypersensitive to LH (15).
Because of its potency, rapid onset of action, and short
half-life, GnRH antagonist quickly and profoundly sup-
presses pituitary FSH/LH secretion. The 0.125 mg/day dose
we used was equally effective as the standard 0.250-mg
dose in preventing premature ovulation. Levels of LH are
lower when the GnRH antagonist is started in the late luteal
phase or with menses rather than on day 6, which suggests
that elevated follicular phase LH levels could be responsible
for the less than anticipated outcomes seen with the standard
GnRH antagonist approach compared with the long agonist
protocol (13, 14). Finally, starting a GnRH antagonist with
menses has been reported to coordinate the growth of the
developing cohort, which may prevent the emergence of
a single dominant follicle (36, 37).
We initially used a GnRH antagonist for its luteolytic ac-
tions to induce menses in women with GnRH agonist–
induced cysts. We continued the GnRH antagonist in these
patients until hCG administration, using a lower dose
(0.125 mg/day) due to the prolonged administration as well
as the recognition that a 2-mg/day GnRH antagonist dose
can completely inhibit ovarian follicular development (38).
In some cases, a suboptimal response may be due to
understimulation. For many, 450 IU/day is considered
the maximum dose of ovarian stimulation (39). However,
we routinely use doses of 600 or 750 IU/day in our
poor-prognosis patients and find benefit in both egg num-
bers and quality over 450 IU/day. The theory is to ensure
all FSH receptors are maximally saturated. We give a sin-
gle daily dose of gonadotropin in an attempt to prevent
receptor down-regulation as a result of persistently high
FSH levels. The risk of overstimulation in these patients
is negligible.
Many patients with anovulatory cycles exhibit androgen
overproduction, which can negatively influence oocyte
development even during ART treatment (40). Because
FSH confers LH sensitivity on granulosa cells before ovula-
tion by inducing LH-receptor formation, an elevated basal
FSH level may induce premature LH-receptor formation.
Estrogen ‘‘priming’’ of FSH receptors has been reported to
slow premature follicular development and to promote gran-
ulosa cell FSH-receptor induction (41, 42). Intramuscular ad-
ministration was chosen over oral or transdermal routes,
which are associated with a high percentage of conversion
to estrone. Estrogen support of the endometrium with vaginal
suppositories was used to maintain the endometrium and to
prevent withdrawal bleeding until an estrogen-producing
follicle has emerged.
Our experience with this protocol over the past 4 years has
given us several insights into the treatment of poor-prognosis
346 Fisch et al. Agonist/antagonist conversion
patients. Although oocyte numbers in a woman may be finite,
oocyte quality in a given cycle can be influenced by the
choice of stimulation medication. Because estrogen domi-
nance in the maturing follicle promotes growth and androgen
dominance promotes apoptosis, the hormonal environment in
the ovary before ovulation is critical to the chance of a given
egg completing meiosis normally.
That there was no difference in ongoing pregnancy rate
with increasing age may indicate a maximized potential in
these patients. More importantly, if each egg has its own
potential to develop normally, prior IVF failure even in the
face of an elevated FSH level or low follicle numbers should
not be used to deny future treatment. Rather, it is a sign that
more careful consideration of the stimulation protocol is
warranted. A larger randomized controlled trial should, of
course, be done to verify these results.
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