Influence of gonadotrophin-releasing hormone agonist total dose in the ovarian stimulation in the long down-regulation protocol for in-vitro fertilization.

Abstract

The goal of our study was to assess whether the total dose of gonadotrophin-releasing hormone agonist (GnRHa) administered affects the success of an in-vitro fertilization (IVF) programme. A retrospective analysis was performed on a total of 72 IVF cycles carried out on 70 patients with different causes of infertility included in our assisted reproduction programme. Cycles were divided into two groups according to the number of days of GnRHa administration (leuprolide acetate 1 mg/day) necessary until ovarian desensitization occurred: group I (GI) < 13 days (n = 27) and group II (GII) > or = 13 days (n = 45). The following parameters were assessed: number of gonadotrophin ampoules, number of stimulation days, endometrial thickness on the day of human chorionic gonadotrophin (HCG) administration, number of recovered oocytes, pregnancy rate. Pregnancy rate/cycle and pregnancy rate/transfer were positively correlated with the dose of GnRHa (GI: 44 and 60% respectively versus GII: 20% and 25% respectively). It is concluded that a long administration of GnRHa has no effect upon ovarian response, although the pregnancy rate is subsequently decreased.

Full text

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Human Reproduction vol.12 no. 11 pp.2366-2369, 1997
Influence of gonadotrophin-releasing hormone agonist
total dose in the ovarian stimulation in the long
down-regulation protocol for in-vitro fertilization
C.Alvarez1, N.Cremades, N.Blasco and R.Bernabeu
Bernabeu Institute, Fertility and Gynecology, 31 Albufereta Avenue,
Alicante 03016, Spain
'To whom correspondence should be addressed
The goal of our study was to assess whether the total
dose of gonadotrophin-releasing hormone agonist (GnRHa)
administered affects the success of an in-vitro fertilization
(IVF) programme. A retrospective analysis was performed
on a total of 72 IVF cycles carried out on 70 patients with
different causes of infertility included in our assisted
reproduction programme. Cycles were divided into two
groups according to the number of days of GnRHa adminis-
tration (leuprolide acetate 1 mg/day) necessary until
ovarian desensitization occurred: group I (GI) <13 days
{n = 27) and group II (Gil) ^13 days in = 45). The
following parameters were assessed: number of gonadotro-
phin ampoules, number of stimulation days, endometrial
thickness on the day of human chorionic gonadotrophin
(HCG) administration, number of recovered oocytes, preg-
nancy rate. Pregnancy rate/cycle and pregnancy rate/
transfer were positively correlated with the dose of GnRHa
(GI: 44 and 60% respectively versus Gil: 20% and 25%
respectively). It is concluded that a long administration of
GnRHa has no effect upon ovarian response, although the
pregnancy rate is subsequently decreased.
Key words: agonists/long down-regulation/total dose
Introduction
Ovarian follicles increase in size under the influence of
gonadotrophins, mainly due to an expansion of follicular fluid
(FF) volume and an acceleration in granulosa cell mitosis. In
a natural cycle, granulosa cells forming the cumulus and
corona layers, and the oocyte that they surround, undergo
synchronous maturational changes. This synchrony may be
disturbed in a gonadotrophin-induced cycle in which interfer-
ence from endogenous luteinizing hormone (LH) may also
cause premature luteinization (Thanki and Schmidt, 1990).
In the treatment of infertility, the transient suppression of
pituitary function can improve the efficacy of gonadotrophin
therapy. The impact of gonadotrophin releasing hormone
(GnRH) on the clinical management of infertility and repro-
ductive endocrinology has been widely reported. Clinical
application of GnRH and its analogues falls into two broad
categories: those dependent upon inhibitory effects on gonado-
trophin secretion and those dependent upon stimulatory effects
of GnRH on gonadotrophin secretion (Gordon et al, 1993).
2366
Gonadotrophin-releasing hormone agonists (GnRHa) are
now used in conjunction with exogenous gonadotrophins as
an integral part of most ovulation induction protocols for
various forms of assisted reproductive technologies (ART).
The principal objective of their use is to reduce the incidence
of premature LH surges (and hence reduce the cancellation
rate) and, by producing a hypogonadotrophic state, to enable
the timing of follicular development to be controlled more
precisely, thereby facilitating scheduling of patients for oocyte
collection (Gordon et al, 1993).
Current clinical practice combines GnRHa treatment with
gonadotrophin therapy. Two stimulation protocols have been
used by different groups: the 'short protocol' combines endo-
genous follicle stimulating hormone (FSH)/LH from the initial
flare effect with exogenous gonadotrophin. The GnRHa treat-
ment usually begins on menstrual cycle day 1 or 2. The 'long
protocol' is often begun in the antecedent mid-luteal phase
(day 19-23) to minimize the flare effect, controlled mainly
by progesterone, oestrogen, and possibly inhibin, from the
functioning corpus luteum (Hodgen, 1990).
The majority of investigators agree over the benefits of
using agonists for ART. However, few IVF groups have
assessed whether the ovarian response to exogenous gonadotro-
phins, cycle performance and pregnancy rate are affected by
different periods of agonist administration in long down-
regulation protocols. In this paper we have attempted to answer
this question.
Materials and methods
Between January and December 1995, a retrospective study was
performed of 72 cycles in 70 patients who were undergoing in vitro
fertilization (IVF) treatment for infertility of diverse aetiology. All
patients were scheduled for IVF-embryo transfer using the long
protocol.
To achieve multiple follicular development we used the following
procedure. Ovarian desensitization was induced by GnRHa (leuprolide
acetate 1 mg/day) in the luteal phase of the preceding cycle. Ovarian
quiescence was verified during the first days of the cycle, by the
absence of follicular growth observed by transvaginal ultrasound, and
oestradiol serum concentration <50 pg/ml. GnRHa administration
ended on the day of human chorionic gonadotrophin (HCG) injection
and ovarian stimulation was started with daily doses of pure FSH
(Neo-Fertinorm; Serono, Madrid, Spain) or FSH combined with
human menopausal gonadotrophin (HMG; Pergonal; Serono). On day
6 of ovarian stimulation the dose was modified according to the
ovarian response observed. The criterion for administering 5000 IU
human chorionic gonadotrophin (HCG; 2500 IU, Profasi; Serono)
was the presence of three or more follicles of ^17 mm in diameter.
Oocyte retrieval was performed 36 h after HCG administration by
transvaginal puncture under ultrasound guidance. These oocytes
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Effects of agonist dose for in-vitro fertilization
were washed with buffer medium (Flushing medium; Medi-Cult,
Copenhagen, Denmark) and classified. All preovulatory oocytes were
preincubated in culture medium (IVF medium; Medi-Cult) for 4—6 h
prior to insemination in an atmosphere of 5% CO2 in air at 37°C.
Sperm concentration and motility were evaluated in a husband's
semen sample. Simple preparative methods such as swim up and
discontinuous Percoll gradients produced satisfactory sperm samples.
Spermatozoa were inseminated in droplets at a concentration of
100 000/ml with normozoospermic samples, a higher concentration
being used with samples from asthenozoospermic and teratozoo-
spermic patients. Inseminated oocytes were cultured overnight (18-
20 h) and examined for two pronuclei and two polar bodies. On the
second day post-insemination, those embryos which had divided to
two or more cells were classified (type 1, 2, 3, 4) and transferred
(three or four embryos/patient) into the uterine cavity. Embryos that
were not used in a fresh transfer were cryopreserved. The luteal
phase was supported with 600 mg/day of progesterone (Utrogestan
100 mg; Laboratoires Besins-Iscovesco, Paris, France). Twelve or 13
days after embryo transfer, the serum fi-HCG concentrations were
determined, a value of >5 mlU/ml being considered to indicate
a positive pregnancy. Pregnancy was confirmed by ultrasound 2
weeks later.
Data are presented as means ± SD and were normally distributed.
Student's f-test was used to discriminate between the two groups, y}
test was used for frequency comparisons. Values of P *£ 0.05 were
considered significant. All statistical evaluations were performed
using the Statistical Package for Social Sciences (SPSS).
Results
Between January and December 1995, 72 cycles were studied
in 70 patients who were undergoing IVF treatment. Cycles
were divided into two groups according to the number of days
of GnRHa administration necessary until ovarian desensitiza-
tion. The median value of 13 days was chosen as the cut-off
Table I. Distribution of patient characteristics and cancelled cycles
GIGil
Cycle number
"Cancelled cycles (%)
Age (years; mean ± SD)
Causes of infertility
Tubal (%)
Male (%)
Unexplained (%)
Endometriosis (%)
Mixed (%)
27
1 (3.7)
32.9 ± 2.0
33.3
22.2
25.9
3.7
14.9
45
2 (4.4)
32.8 ± 2.8
33.3
24.4
22.3
4.4
15.6
point: in group I (27 cycles), the down-regulation period before
controlled ovarian hyperstimulation (COH) was <13 days,
and in group II (45 cycles) the period was 5=13 days.
The following parameters were determined: number of
gonadotrophin ampoules, number of stimulation days, endo-
metrial thickness on the day of HCG administration, the mean
number of oocytes recovered and pregnancy rate.
Patients in the two groups were similarly distributed in age
(32.9 ± 2.0 years versus 32.8 ± 2.8 years) and cause of
infertility (Table I). Two patients (4.4 %) had ovarian cysts in
group II. The number of cycles cancelled because of poor
ovarian response was similar between the two groups (3.7
versus 4.4%) (Table I).
The amount of exogenous gonadotrophins required, length
of the stimulation period, endometrial thickness on the day of
HCG administration and the mean number of oocytes collected
were very similar in the two groups (Table II). No statistically
significant differences were found.
There was a relationship between the number of days of
GnRHa administration and the clinical pregnancy rate. The
pregnancy rate by transfer was significantly lower in the
patients group suppressed for a longer period (group II) (P <
0.05; see Table II).
Discussion
The last few years have seen the increasing use of GnRHa
during controlled ovarian hyperstimulation for ART. Today
the majority of investigators agree over the benefits of using
agonists, the main medical advantage consisting of the reduc-
tion of the rates of cancellation due to poor responses or to
premature luteinization (Hughes et al, 1992; Calhaz Jorge
et al, 1995; Senoz et al, 1995; Devreker et al, 1996; Ferrareti
et al., 1996). Other reports have indicated that GnRHa protocols
might also establish a good uterine environment, although
flare-up regimens have been associated with poor rates of
fertilization and low embryo quality. Many authors advocate
the use of long GnRHa protocols to obtain a complete down-
regulation of pituitary gland, to establish basal conditions in
the ovary, and to facilitate the programming of cycles (Edwards
and Brody, 1995).
It is well known that desensitization protocols require
administration of an agonist for several days to achieve
consistent suppression of ovarian activity. In our study, we
demonstrated that higher doses of GnRHa (the degree of
pituitary suppression) do not affect the response to stimulation.
Table II. Ovarian stimulation results and pregnancy rate
Ampoules of
gonadotrophin
Days stimulation
Endometrial
thickness
Oocyte recovery
Pregnancy
rate/cycle (%)
Pregnancy
rate/transfer
GI
n = 27
Gil
n = 45
36.0 ± 9.6
38.0±7.8
9.0
10.0
± 1.9
±1.6
11.2
11.4
± 1.9
± 2.7
13.5
11.8
± 7.1
± 5.0
44*
20*
60*
25*
Results are expressed as mean ± SEM.
n = cycle number.
* Statistically significant differences were found among the groups (P < 0.05).
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CAlvarez et al.
When the number of days of agonist administration
was increased, despite inhibition of endogenous pituitary
gonadotrophin secretion by GnRHa, the number of stimulation
days and the number of gonadotrophin ampoules did not
differ from those of patients with fewer administration days
(Neven et al, 1987). Similar amounts of exogenous
gonadotrophins and similar duration of treatment yielded
the same number of oocytes recovered from both groups,
in agreement with results that have been described previously
in more detail (Calhaz Jorge et al, 1995; Senoz et al,
1995; Ferrareti et al, 1996). Olivennes et al. (1996) observed
a better ovarian response and higher quality embryos in
patients with a high basal FSH concentration on day 3
when they were treated with a low dose of GnRHa. In our
study, it is possible that patients who needed a lower dose
of GnRHa to produce suppression of ovarian activity had
a high basal FSH concentration. However, this cannot be
confirmed because we lack these data. On the other hand,
in our IVF programme, basal FSH concentrations were
obtained in patients with cycles previously cancelled due to
poor response. In this group of patients, ovarian stimulation
was achieved using the 'short protocol'.
Other reports have shown decreased number and quality
of human oocytes and embryos obtained after exaggerated
follicular stimulation using high doses of GnRHa (Testart
et al, 1986; Balasch et al, 1993). In addition, expression
of GnRH receptor messenger RNA in human ovaries has
been demonstrated, providing evidence that the ovary may
be a target for extrapituitary GnRH action (Minaretzis et al,
1995). Moreover, it has been proved (Devreker et al, 1996)
that the analogue is able to diffuse into FF. With long-
acting GnRHa, larger numbers of molecules persist in the
blood circulation far beyond the injection of HCG and
oocyte retrieval. The presence of GnRHa in the FF during
oocyte maturation may interfere with embryo metabolism,
impairing the potential of the future embryo to implant and
giving rise to abnormalities that do not manifest themselves
morphologically (Devreker et al, 1996).
The comparison between IVF-embryo transfer cycles is
complicated by differences between stimulation treatments.
Different protocols have been designed for the administration
of GnRH together with gonadotrophins for ovarian stimulation
(Loumaye, 1990). One parameter subject to variation is the
duration of GnRHa administration before starting the ovarian
stimulation with gonadotrophins. Ultrashort, short and long
protocols have been tested and overall results in the literature
indicate that the long GnRHa protocol is associated with a
higher pregnancy rate (Loumaye, 1990; Tan et al, 1992).
A second parameter subject to variation is the time of
initiation of GnRHa therapy. The timing of GnRHa
administration during the menstrual cycle may influence the
time course of ovarian suppression and the IVF outcome
(Ferrareti et al, 1996). Moreover, different patterns of
follicular growth were observed in humans according to the
use of different GnRH molecules (buserelin, triptorelin or
leuprorelin) (Testart et al, 1993). These variations may
explain discrepancies between our findings and those reported
previously concerning the success of IVF. Clinical studies
do not permit the identification of a direct effect of GnRHa
either on the oocyte or on the uterus, since such effects
are cumulative in the results of IVF-embryo transfer. Only
research studies could demonstrate specific effects of GnRHa
molecules on ovaries, oocytes and embryo implantation.
On the other hand, the endometrial thickness on the day
of HCG administration was very similar in both groups,
despite the remarkably higher pregnancy rate in group I.
Although we observed a similar number of cancelled
cycles in both groups, our sample size is too small to
demonstrate a significant difference between the two groups.
The present results show that a longer period of GnRHa
administration to achieve ovarian desensitization has no
effect on the ovarian response to gonadotrophin stimulation,
although it leads to a decrease the clinical pregnancy rate
(Neyro et al, 1994). This may provide further information
on the effect of long-acting GnRHa on uterine receptivity.
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