Human Reproduction vol.13 no.3 pp.525–530, 1998
Depression of FSH and LH secretion following pulsatile
GnRH administration in ovariectomized women
Marı́a Graña-Barcia1,4, Joaquı́n Lado-Abeal2,
José-Luis Liz-Lestón1, Santiago Lojo3,
Alejandro Novo-Domı́nguez1 and
Jaime Aguilar-Fernández1
1Department
of Obstetrics and Gynaecology and 2Endocrinology
Service and 3Central Laboratory, University Hospital Complex,
School of Medicine, Galeras s/n, 15705 Santiago de Compostela,
Spain
4To
whom correspondence should be addressed
To investigate the mechanism by which pulsatile administration of gonadotrophin-releasing hormone (GnRH)
modifies secretion of luteinizing hormone (LH) and
follicle-stimulating hormone (FSH), we studied three
groups of five women who had been ovariectomized for
non-malignant gynaecological conditions at least 6 months
previously, none of whom had received substitutional
hormone therapy. Before and after 15 day treatment
with subcutaneous pulsatile GnRH (one 20 µg dose every
90 min in group A, one 10 µg dose every 90 min in
group B and one 20 µg dose every 120 min in group
C), pulsatile secretion of LH and FSH was characterized
by determining these hormones in 4 ml blood samples
taken every 10 min for 8 h (9.00 a.m. to 5.00 p.m.). For
both LH and FSH, mean serum concentration and pulse
amplitude were lower after GnRH treatment than before
(and in the case of LH the decrease depended upon both
the size and frequency of exogenous GnRH pulses) but
in no group was there a significant change in LH or
FSH pulse frequency. We conclude that exogenous
pulsatile GnRH probably acts by partially desensitizing
the pituitary rather than by depressing endogenous
GnRH secretion. Such partial desensitization would
explain reports that exogenous pulsatile GnRH improves
ovulation by women with polycystic ovary syndrome.
Key words: exogenous pulsatile GnRH/GnRH analogues/
induction of ovulation/IH and FSH rhythms/pituitary desensitization
Introduction
The amplitude and frequency of the gonadotrophin pulses
secreted by the pituitary of women of fertile age depend on
the phase of the menstrual cycle (Filicori et al., 1986). Secretion
is stimulated by pulses of gonadotrophin-releasing hormone
(GnRH) that are emitted by the hypothalamus under the control
of the ‘GnRH pulse generator’ and other signals relayed via
diverse neuron nuclei (Leranth et al., 1992), and is regulated
by feedback from the gonads.
© European Society for Human Reproduction and Embryology
We have previously reported that pulsatile subcutaneous
administration of GnRH induces ovulation more rapidly in
women with low or zero gonadotrophin levels due to hypogonadotrophic hypogonadism of hypothalamic origin, than in
women with normal gonadotrophin levels receiving GnRH for
oligomenorrhoea (Graña, 1997). We interpreted this finding as
showing that exogenous GnRH requires a certain time to
prevail over the action of endogenous GnRH. Two suggestions
have been put forward as to the mechanism by which exogenous
GnRH prevails over the endogenous hormone: partial desensitization of the pituitary (Lambalk et al., 1986, 1987) and
depression of secretion of GnRH by the hypothalamus
(Lambalk et al., 1991).
To clarify the mechanism of action of exogenous pulsatile
GnRH, we have now studied the effects of three different
administration regimens on secretion of follicle stimulating
hormone (FSH) and luteinizing hormone (LH) in ovariectomized women.
Material and methods
Subjects
We studied 15 women aged 44–55 years who had been ovariectomized on account of non-malignant gynaecological conditions at
least 6 months previously (seven for uterine leiomyoma, four for
ovarian endometriosis and four for ovarian cystadenoma). None
had received substitutional hormone treatment, and none had taken
psychotropic, anti-epileptic or neuroleptic drugs in the 3 months
prior to this study. All volunteered to take part in the study after
being duly informed of its nature, and gave written consent.
Before their inclusion, determination of FSH, LH, oestradiol,
triiodothyronine (T3), thyroxine (T4) and thyroid-stimulating hormone (TSH) confirmed surgical menopause (FSH .40 IU/l,
oestradiol ,50 pg/ml) and ruled out thyroid dysfunction and other
alterations, and standard haematological and biochemical parameters
were found to be normal.
Experimental protocol
The subjects were assigned to three groups: the first five to group A,
the second five to group B and the third five to group C. In each
subject, pulsatile secretion of LH and FSH was characterized before
(day 0) and after (day 16) a group-specific 15 day treatment with
subcutaneous pulsatile GnRH (Luforan® 500, Serono, Madrid, Spain),
administered by means of a Zyklomat Pulse infusion pump (Ferring,
Kiel, Germany). Group A subjects received one 20 µg dose every
90 min, group B subjects a 10 µg dose every 90 min and group C
subjects a 20 µg dose every 120 min. Characterization of LH and
FSH secretion was based on their determination in 4 ml blood samples
taken every 10 min over an 8 h period (09.00–17.00 h), during which
time the subject remained seated or supine and took a continental
breakfast and a light lunch, together totalling approximately 2000
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M.Graña-Barcia et al.
Figure 1. Serum LH (s-s) and FSH (d-d) concentrations during the pre-treatment (left) and post-treatment (right) studies in subjects from
groups A (top), B (middle) and C (bottom). The pulses detected are marked as arrows (↓).
calories. The last GnRH pulse was administered 1 min before the
third blood sample of the post-treatment characterization session
was taken.
Determination of hormones
Blood samples were centrifuged immediately and the resulting sera
were stored at –20°C pending analysis. FSH, LH and oestradiol were
determined in triplicate (in each case with the three subsamples in a
single run) using an ACS-180 Plus automatic analyser (Chiron
Diagnostics, Medfield, MA, USA) and reagents from the same
supplier; FSH and LH were determined by sandwich immunoluminescence assays, and oestradiol by a competitive immunoluminescence
assay using monoclonal antibody against position 6 of the oestradiol
molecule. The FSH method, calibrated with WHO 2nd International
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Reference Preparation (1980) (NIBSC 78/549), had a detection limit
of 0.03 IU/l and conditional coefficients of variation (CVs) of 6.81%
at 5.9 IU/l and 6.35% at 64.7 IU/l. The LH method, calibrated with
WHO 2nd International Reference Preparation (1988) (NIBSC 80/
552), had a detection limit of 0.09 IU/l and conditional CVs of 3.86%
at 5.5 IU/l and 4.14% at 36.1 IU/l.
Pulse detection
LH and FSH pulses in serum were detected by a non-parametric
statistical method (Lado-Abeal et al., 1991) that in validatory trials
with simulated LH series had achieved a sensitivity and positive
accuracy (Urban et al., 1989) of 0.88 and 1.00 respectively, with a
sum of 1.88 similar to that reported for the cluster method (Urban et al.,
1988). The parameters calculated were pulse amplitude, interpulse
Exogenous pulsatile GnRH desensitizes the pituitary
Table I. Mean serum LH concentrations (IU/l)a in three groups of five ovariectomized women before and
after 15 days pulsatile exogenous GnRHb, and the median of the percentage fall in mean serum LH
concentration in each group
Group
Before
After
P value
Fall in LH (%)
Group A
Group B
Group C
22.12 6 3.77 (27.10)
36.88 6 19.03 (29.80)
22.17 6 2.22 (21.95)
A versus B versus C
NS
5.74 6 1.94 (5.40)
13.72 6 5.71 (13.00)
8.80 6 0.59 (8.70)
A versus B P , 0.05
A versus C P , 0.05
B versus C NS
, 0.001
, 0.001
, 0.001
75.00
52.03
62.64
A versus B P , 0.05
A versus C P , 0.05
B versus C NS
6 SD with medians in parentheses.
A, one 20 µg pulse every 90 min; group B, one 10 µg pulse every 90 min; group C, one 20 µg
pulse every 120 min.
NS 5 not significant.
aMeans
bGroup
Table II. LH pulse amplitude (IU/l)a and interpulse intervals (min)
Group
Amplitudes
Group A
Group B
Group C
Interpulse intervals
Group A
Group B
Group C
aMeans
Before
After
P value
4.52 6 2.25 (4.60)
5.55 6 2.53 (5.50)
5.58 6 2.55 (5.65)
A versus B versus C
NS
1.25 6 0.74 (1.10)
3.04 6 2.69 (2.10)
2.33 6 1.27 (1.91)
A versus B P , 0.001
A versus C P , 0.001
B versus C NS
, 0.001
, 0.001
, 0.001
56.41 6 17.55 (60)
63.44 6 25.60 (60)
71.29 6 24.19 (70)
A versus B NS
A versus C P , 0.05
B versus C NS
60.69 6 19.81 (60)
62.65 6 26.32 (60)
74.61 6 25.96 (70)
A versus B NS
A versus C P , 0.05
B versus C NS
(NS)
(NS)
(NS)
6 SD with medians in parentheses; NS 5 not significant.
Table III. Mean serum FSH concentrations (IU/l)a and the median of the percentage fall in mean serum FSH
concentration in each group
Group
Before
After
P value
Fall in FSH (%)
Group A
Group B
Group C
58.44 6 20.26 (67.90)
59.50 6 19.22 (64.00)
38.90 6 7.51 (37.70)
A versus B versus C
NS
35.47 6 13.42 (40.35)
38.14 6 18.09 (32.70)
29.86 6 5.48 (27.80)
A versus B versus C
NS
, 0.001
, 0.001
, 0.001
34.90 %
44.70 %
23.42 %
A versus B versus C
NS
aMeans
6 SD with medians in parentheses; NS 5 not significant.
interval, the number of pulses in the 8 h session, and mean hormone
level over the 8 h period.
Expression of results and statistical analysis
Pulse amplitude, interpulse interval and mean concentration results
are expressed in the tables below as means 6 SD for each group and
session, with medians in parentheses. The statistical significance of
differences among groups was estimated with the Kruskal–Wallis
test; when significant differences were detected, pairwise comparisons
were effected using the Mann–Whitney test. Within-group differences
between pre- and post-treatment values were assessed with the
Wilcoxon matching pairs test.
Results
Figure 1 shows how serum LH and FSH concentrations varied
during the pre-treatment and post-treatment studies in three
subjects (one from each group).
Mean serum LH concentration
During the first (pre-treatment) evaluation, there were no
statistically significant differences among the mean serum LH
concentrations in the three subject groups (Table I). The mean
levels in the post-treatment session were decreased. The fall
was significantly larger for group A than for groups B and C,
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M.Graña-Barcia et al.
Table IV. FSH pulse amplitude (IU/l)a and interpulse intervals (min)
Group
Amplitudes
Group A
Group B
Group C
Interpulse intervals
Group A
Group B
Group C
aMeans
Before
After
P value
7.67 6 5.19 (5.40)
5.95 6 3.04 (5.80)
4.23 6 2.15 (4.20)
A versus B NS
A versus C P , 0.01
B versus C P , 0.05
3.48 6 2.49 (2.70)
6.65 6 6.87 (4.60)
2.88 6 1.51 (2.80)
A versus B versus C
NS
, 0.001
NS
, 0.05
62.19 6 49.30 (40)
88.33 6 55.92 (80)
86.25 6 40.09 (80)
A versus B P , 0.05
A versus C P , 0.01
B versus C NS
66.67 6 43.35 (55)
67.74 6 41.37 (60)
85.00 6 45.40 (85)
A versus B versus C
NS
NS
NS
NS
6 SDs with medians in parentheses.
in keeping with which the post-treatment value for group A
was significantly lower than those for groups B and C, which
did not differ significantly from each other.
LH pulses
Pre-treatment, there were no significant differences among the
three groups as regards LH pulse amplitude (Table II). The
post-treatment values were significantly lower than the pretreatment values in all three groups, and were significantly
lower in group A than in group B or group C. Both pre- and
post-treatment, the interval between LH pulse peaks was
significantly shorter in group A than in group C, but in no
group was there a significant difference between pre- and posttreatment values (Table II).
Mean serum FSH concentration
In each group, mean serum FSH concentration was significantly
lower after GnRH treatment than before, but neither before
nor after treatment were there significant differences among
the three groups (Table III).
FSH pulses
Pre-treatment FSH pulse amplitude was significantly smaller
in group C than in the other groups, but the three groups did
not differ significantly in this respect after treatment (Table IV).
Post-treatment FSH pulse amplitude was only significantly
different from the pre-treatment value in the groups receiving
20 µg GnRH pulses. Before GnRH treatment, the interval
between FSH pulse peaks was significantly shorter in group
A than in group B or group C. For no group was the posttreatment FSH interpulse interval significantly different from
the pre-treatment value, nor were there any significant differences in post-treatment FSH interpulse interval among the
three groups.
Discussion
Menopausal and ovariectomized women are ideal subjects
for the study of pituitary gonadotrophin secretion and its
modification because of their large FSH and LH pulses and
528
the absence of ovarian feedback to which these large pulse
amplitudes are due.
In this study, 15 day subcutaneous pulsatile administration
of GnRH to ovariectomized women was followed by a fall in
their mean serum LH concentrations and LH pulse amplitudes
to values typical of the medium-late follicular phase (LadoAbeal et al., 1991). The extent of the reduction depended in
both cases on both the size and the frequency of the GnRH
pulses, the most effective of the three regimens used being
one 20 µg dose every 90 min. These findings agree with those
of other authors (Uemura et al., 1992; Scheele et al., 1996).
The GnRH treatment was also followed by a fall in mean
serum FSH concentrations, but the percentage reduction was
less than for LH and did not differ significantly among groups
(which is compatible with the possibility of a mechanism that
is independent of GnRH pulse size and frequency). FSH pulse
amplitude was significantly reduced by administering GnRH
at a level of 20 µg per pulse, regardless of pulse frequency,
but not by 10 µg pulses.
The intervals between gonadotrophin pulses were not significantly altered by the GnRH treatment in any group. This
suggests that the GnRH-induced reduction in gonadotrophin
levels is less likely to have been due to suppression of
hypothalamic emission of GnRH (one of the mechanisms
mooted by Lambalk et al., 1991) than to partial desensitization
of the pituitary by GnRH, a well-documented effect of exogenous GnRH (Lambalk et al., 1986; Conn and Crowley, 1991).
The differences between the responses of LH secretion and
FSH secretion to exogenous pulsatile GnRH suggest that
synthesis and/or release of FSH may not be so dependent on
GnRH as in the case of LH, an observation previously made
by Genazzani and co-workers (Genazzani et al., 1994, 1996).
This would explain why GnRH analogues reduce LH and FSH
levels to markedly different extents, inhibiting LH secretion
almost completely but reducing serum FSH only to medium
follicular phase levels (Broekmans et al., 1993).
There is controversy as to whether the use of GnRH
analogues is always advantageous in reproductive medicine
(Kingsland et al., 1992) or guarantees a reduction in the
incidence of serious complications of gonadotrophin treatment
Exogenous pulsatile GnRH desensitizes the pituitary
such as multiple pregnancy or ovarian hyperstimulation syndrome (OHS) (Ron-El et al., 1991). It has been suggested that
GnRH analogues may even favour the development of OHS,
especially in women with polycystic ovary syndrome (PCOS)
(Navot et al., 1992), and that they may harm the oocyte
(Pellicer et al., 1992; Testart et al., 1993). PCOS involves high
serum LH concentrations (Burger et al., 1989) due to both the
frequency and amplitude of LH pulses being increased by a
hyperactive hypothalamo–pituitary axis (Waldstreicher et al.,
1986; Kazer et al., 1987), but it seems plausible that to treat
PCOS and other anovulatory conditions involving high LH
levels, it may suffice to depress pituitary activity partially
rather than entirely (Monroe et al., 1986; Filicori et al., 1993).
The partial suppression of LH and FSH levels in the present
study of ovariectomized women suggests that this can be
achieved by pulsatile GnRH treatment, and this notion seems
to be in consonance with reports that the ovulation of women
with PCOS is improved by pulsatile administration of GnRH
both when this treatment is given following administration of
GnRH analogues (Filicori et al., 1991) and also when it is
given in successive menstrual cycles (Corenthal et al., 1994).
We postulate that in both cases pulsatile GnRH causes partial
suppression of gonadotrophin secretion: when given following
GnRH analogues, this results in its being associated with
an immediate rise in gonadotrophin levels (given the total
suppression brought about by the analogues), and when given
in successive cycles it brings about a fall in gonadotrophin
levels (an effect that only becomes manifest in the second
cycle). It is therefore possible that the best GnRH ‘analogue’
is exogenous GnRH, in the sense that GnRH achieves the
desired effects without completely suppressing LH secretion
and without giving rise to climacteric symptoms.
The dependence of follicle maturation on the presence of
low levels of LH as well as FSH has recently been stressed
by Hillier (1996), who stated that in women with WHO
Type 1 infertility, the response to pure FSH will depend on
simultaneous administration of LH. The induction of pituitary
production of both gonadotrophins by pulsatile exogenous
GnRH may be a more natural alternative to direct administration of gonadotrophins.
To sum up, in this work we found that 15 day subcutaneous
pulsatile administration of GnRH to ovariectomized women
brought about a significant dose- and frequency-dependent
reduction in the mean serum concentration and pulse amplitude
of LH, together with a reduction in mean FSH levels that, in
this study, did not depend on dose or frequency to a statistically
significant extent; the intervals between LH or FSH pulses
were not altered by the GnRH treatment. We conclude that
pulsatile administration of GnRH at an appropriate pulse
frequency probably reduces gonadotrophin levels by decreasing
the sensitivity of the pituitary to GnRH rather than by
depressing hypothalamic GnRH emission, and reason that this
effect may prove useful for hormonal control in reproductive
medicine.
Acknowledgements
We thank Ma Isabel Fernández-Jiménez, Ma Carmen Crecente Paredes
and Angeles Sanmartı́n-López for technical assistance. This work
was funded in part by the Xunta de Galicia under Project XUGA
20804B95.
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Received on March 14, 1997; accepted on November 27, 1997
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