Download Pure and highly purified follicle-stimulating hormone alone or in

Human Reproduction vol.11 no 11 pp 2400-2404, 1996
Pure and highly purified follicle-stimulating hormone
alone or in combination with human menopausal
gonadotrophin for ovarian stimulation after pituitary
suppression in in-vitro fertilization
Juan Balasch1, Francisco Fabregues,
Montserrat Creus, Vicenta Moreno,
Bienvenido Puerto, Joana Peflarrubia,
Francisco Carmona and Juan A.Vanrell
Department of Obstetrics and Gynecology, Faculty of MedicineUniversity of Barcelona, Hospital Clinic i Provincial, Barcelona,
Spain
'To whom correspondence should be addressed at: Department of
Obstetrics and Gynaecology, Hospital Clinic i Provincial,
C/Casanova 143, 08036-Barcelona, Spain
The use of pure follicle stimulating hormone (pFSH) and
highly purified FSH (FSH-HP) versus the combinations
pFSH/human menopausal gonadotrophin (HMG) and FSHHP/HMG, respectively, was compared for stimulating follicular development after gonadotrophin-releasing hormone
agonist (GnRHa) suppression in women undergoing invitro fertilization (TVF)-embryo transfer. Two consecutive
prospective, randomized studies were carried out at
the Assisted Reproduction Unit of the Hospital Clinic i
Provincial in Barcelona, a tertiary care setting. Two groups
of 188 (study 1) and 252 (study 2) consecutive infertile
patients respectively, scheduled for IVF-embryo transfer
were included. Pretreatment with leuprolide acetate (long
protocol) was followed by gonadotrophin treatment in all
patients. In study 1, 92 patients received i.m. pFSH alone
(group pFSH) and 96 were treated with the combination
of i.m. pFSH and i.m. HMG (group HMG-1). In study 2,
123 patients received s.c. FSH-HP alone (group FSH-HP)
and 129 patients were given the combination of s.c FSH-HP
and i.m. HMG (group HMG-2). Main outcome measures
included follicular development, oocyte retrieval, fertilized
oocytes, duration and dose of gonadotrophin therapy, and
clinical pregnancy. There were no significant differences
between pFSH and pFSH/HMG nor between FSH-HP
and FSH-HP/HMG cycles with regard to the number of
ampoules of medication used, day of human chorionic
gonadotrophin (HCG) administration, mean peak serum
oestradiol concentrations, number of follicles punctured,
and number of oocytes aspirated, embryos transferred, or
pregnancies. We conclude that urinary FSH (either purified
of highly purified) alone is as effective as the conventional
combination of urinary FSH/HMG for ovarian stimulation
under pituitary suppression in IVF cycles. Therefore, they
can be used interchangeably in FVF programmes.
Key words: highly purified FSH/TVF/ovarian stimulation/pure
FSH/urofollitrophins
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Introduction
Pituitary desensitization with the long protocol increases the
probabilities of conception in in-vitro fertilization (IVF)embryo transfer (Hughes et al, 1992; Tan et al, 1994) and it
was found that the choice of gonadotrophin-releasing hormone
agonist (GnRHa) may influence results obtained (Balasch et al,
1992; Penzias et al, 1992). To stimulate multiple follicular
development for IVF-embryo transfer treatment, most IVF
centres have used the usual equal mixture of follicle stimulating
hormone (FSH) and luteinizing hormone (LH) in the form of
human menopausal gonadotrophin (HMG) alone or adding
purified FSH on the first 2 days of HMG stimulation (Strickler
et al, 1995). This is based on traditional ovulation induction
in cases of amenorrhoea due to hypothalamic-pituitary failure
and gonadotrophin deficiency and on the belief that stimulation
of the ovaries under pituitary suppression would require
supplementation with LH (Hull et al., 1994). However, the
concept is emerging that 'resting' concentrations of LH,
eventually acting synergistically with locally produced growth
factors, are sufficient for oocyte maturation and steroidogenesis. Furthermore, in women with normal pituitary function,
treatment with FSH alone after pituitary suppression of endogenous gonadotrophin production with a GnRHa, results in
normal increases of oestradiol, ovulation and fertilization,
showing that the amount of LH that escapes the pituitary
down-regulation is sufficient (Hillier, 1994; Simoni and
Nieschlag, 1995).
On the basis of the above evidence, different authors
have carried out comparisons between 'pure' FSH (pFSH,
urofollitrophin) and HMG in IVF cycles and a recent metaanalysis of these data indicates that the overall odds ratio (OR)
for the eight trials analysed favours the use of FSH which
results in >50% improvement over HMG in clinical pregnancy
rates (Daya et al, 1995). However, the individual ORs for
each trial were not statistically significant and all but one of
these previous studies only included around 20 patients in
each group of patients. In addition, two more recent reports
comparing GnRHa-HMG and GnRHa-highly purified FSH
(FSH-HP) protocols concluded that FSH-HP resulted in
reduced concentrations of serum oestradiol as compared to
HMG cycles, but some beneficial effects of HMG on the
number of oocytes retrieved, fertilization rates and pre-embryo
development were demonstrated (Fried et al., 1996;
Westergaard et al, 1996). Also it should be noted that none
of those previous reports compared urinary FSH alone with
the combination of urinary FSH and HMG (days 1 and 2 of
ovarian stimulation) followed by HMG, nor used FSH-HP.
Thus, further studies are necessary to optimize the gonadotrophin regimen in IVF.
© European Society for Human Reproduction and Embryology
FSH versus FSH/HMG after GnRHa
In our FVF programme ovanan stimulation is usually carried
out using urinary FSH and HMG (Balasch et al, 1992). The
objective of the present report was to compare the efficacy of
this standard combined therapy with pFSH alone and FSH-HP
alone for stimulating multiple ovanan folhcular development in
women undergoing IVF-embryo transfer.
Materials and methods
The present report shows the results of two successive randomized,
prospective studies including IVF patients. Study 1 was earned out
to compare pFSH alone with pFSH/HMG for ovarian stimulation
under pituitary suppression with GnRHa, while in study 2, FSH-HP
alone was compared with FSH-HP/HMG. The study populations
consisted of two groups of 188 (study 1) and 252 (study 2) consecutive
patients respectively, scheduled for IVF-embryo transfer. All women
gave informed consent to be included in the study which was approved
by the Ethics Committee of our department All of them were
premenopausal women aged 23-39 years and had no ovarian failure
according to FSH concentrations <12 mlU/ml (standard ERP 78/
549) As previously reported (Balasch et al., 1992), in our IVF
programme ovarian stimulation is routinely accomplished using
gonadotrophin treatment under pituitary suppression with GnRHa. In
all women, leuprolide acetate (Procnn. Abbott Laboratories S.A ,
Madrid, Spain) suppression was started in the mid-luteal phase of the
previous cycle at a daily dose of 1 mg. It was continued for 13-15
days prior to treatment with gonadotrophins. That dose was reduced
to 0.5 mg daily once ovanan arrest was achieved and then continued
until the administrauon of human chorionic gonadotrophin (HCG)
Gonadotrophic stimulation of the ovaries was started when serum
oestradiol concentrations declined to <50 pg/ml and a vaginal
ultrasonographic scan showed an absence of follicles >10 mm
diameter.
For the specific purpose of the present investigation patients were
allocated to a gonadotrophin treatment group according to a computergenerated randomization table. In study 1, 92 patients received l.m.
pFSH alone (group pFSH) and 96 were treated with the combination
of i.m. pFSH and l.m. HMG (group HMG-1). In study 2, 123 patients
received s c. FSH-HP alone (group FSH-HP) and 129 patients were
given the combination of s.c FSH-HP and i.m HMG (group HMG-2).
The scheme of gonadotrophin administration was the same in the
four groups studied. On days 1 and 2 of ovanan stimulation three
ampoules per day of HMG (Pergonal: Serono S.A., Madnd, Spain)
were administered together with three ampoules of either pFSH
(Fertinorm: Serono S.A ) (group HMG-1) or FSH-HP (Neo-Fertinorm:
Serono S.A.) (group HMG-2). Patients included in groups pFSH
and FSH-HP were given six ampoules daily of these respective
gonadotrophin preparations on days 1 and 2 of ovulation induction
therapy. On days 3-7 of ovarian stimulation, two ampoules per day
of HMG (groups HMG-1 and HMG-2), pFSH (group pFSH) or FSHHP (group FSH-HP) were administered to each patient From day 8
onward, each gonadotrophin preparation was administered on an
individual basis according to the ovarian response Transvaginal
ultrasonography and serum oestradiol measurement were performed
daily from day 7 of ovarian stimulation to assess folhcular development The cntena for HCG administrauon (5000 IU, Profasi: Serono
S.A.) were the presence of two or more follicles >17 mm in diameter
and oestradiol concentrations >800 pg/ml (conversion factor to SI
units, 3.671).
Oocyte aspiration was performed by vaginal ultrasonography 35-37
h after HCG administration under local anaesthesia. The maturauonal
status of the oocytes was recorded according to the cntena of Veeck
(1986). Up to four embryos were replaced and the remaining were
cryopreserved. Additional doses of 5000, 2500, and 2500 IU HCG
were given on the day of follicle aspiration and 2 and 5 days later,
respectively, to supplement the luteal phase in all patients. In pauents
with serum oestradiol concentrations >4000 pg/ml, HCG support
was withheld to reduce the nsk of ovarian hyperstimulation syndrome
Instead, these patients were given micronized progesterone administered intravaginally at a dose of 600 mg/day (given at 8 h intervals)
from the day of embryo transfer until menses nr serological confirmation of pregnancy.
Oestradiol concentrations in serum were estimated by direct radiolmmunoassay (BioMeneux, Marcy I'Etoile, France). Intra-assay and
inter-assay coefficients of variation were < 5 and <5.8% respectively
Ultrasonic scans were performed using a 5 MHz vaginal transducer
attached to an Aloka sector scanner (Model SSD-620: Aloka Co. Ltd,
Tokyo, Japan).
Because the aim of ovanan stimulation is to increase the number
of oocytes available for fertilization (Hedon et al, 1995), the sample
size (>90 patients/group) was calculated assuming a power of 80%
to detect a difference of 20% between groups in the number of
oocytes retrieved, with a type I risk of 0.05. Statistical comparisons
were performed by cm-square analysis or Student's /-test as appropnate Significance was defined as P < 0 05
Results
Results are summarized in Tables I-UI. From Table I it can
be seen that main demographic and baseline characteristics of
the patients in groups pFSH and HMG-1 (study 1) and in
groups FSH-HP and HMG-2 (study 2) were similar in terms
of age, body mass index (BMT), cause of infertility, duration
of infertility, early folltcular phase FSH, and number of IVF
attempts. In addition, no significant differences regarding these
parameters were found when groups pFSH versus FSH-HP
and HMG-1 versus HMG-2 respectively, were compared. This
supports the validity of the randomization process.
The mean time for total ovarian arrest, days of ovanan
stimulation, the total dose of gonadotrophins, follicular recruitment and growth during gonadotrophin treatment, and the
endometrial thickness and oestradiol serum concentrations on
the day of HCG administration, were similar for the two
groups in study 1 and for the two groups in study 2 (Table H).
Also, there were no significant differences between groups
pFSH and FSH-HP nor groups HMG-1 and HMG-2 with
respect to those parameters of ovarian response.
Regarding ovum retrieval and IVF outcome, the number of
follicles punctured, the number and quality of oocytes retrieved,
the number of fertilized oocytes, the number of embryos
suitable for replacement and cryopreservation, and the number
of patients with embryo transfer, were compared between
groups in studies 1 and 2 (Table III). No significant differences
were found for any of these variables in both studies. Again,
results were similar when group pFSH versus FSH-HP and
HMG-1 versus HMG-2 respectively, were compared.
The combination of pFSH or FSH-HP with HMG and
treatments with pFSH alone or FSH-HP alone were found
not to be different when the number of clinical pregnancies
as well as the pregnancy rate per initiated cycle, per ovum
retrieval procedure, and per embryo transfer were compared.
The number of losses due to spontaneous abortion was
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J.Balasch et aL
Table L Main demographic and baseline characteristics of patients
Study 1
Study 2
Variable*
pFSH (n = 92)
HMG-1 (n = 96)
FSH-HP (n = 123)
HMG-2 (n = 129)
Age (years)b
BM1 (kg/m2)b
Cause of infertility (n,%)
Tubal
Unexplained
Endometriosis
Male
Duration of infertility (years)b
Days 2-A FSH (mIU/ml)b
No. of IVF attempts'1
33.8 ± 3.0
23.3 = 2 4
34.1 ± 3 6
23.6 ± 2 2
33 3 ± 3 3
228 ± 22
33.7 ± 3 3
23 5 ± 2 6
46(50)
18 (19)
15 (16)
13 (14)
7.0 ± 3 5
7.0 ± 2 9
1 7 ± 1.0
46(48)
22 (23)
11(11)
17 (18)
6 6 ± 3.5
6 7 ± 2.7
1.5 ± 1.0
49(40)
25(20)
27 (22)
22 (18)
62 ± 27
6 8 ± 3.2
15 + 0 8
55 (42)
30(23)
25(19)
19 (15)
66 ± 3 1
7.2 ± 29
1 7 ± 0.9
•No significant differences between pFSH and HMG-1 groups nor between FSH-HP and HMG-2 groups
•Values are means ± SD.
BMI = body mass index, FSH =• follicle stimulating hormone, pFSH = pure FSH, FSH-HP = highly purified FSH, HMG = human menopausal
gonadotrophin, IVF = ln-vitro fertilization.
Table EL Ovarian response m the four groups of IVF patients
Study 1
1
Variable
b
Time for ovarian arrest (days)
Total no of ampoules of gonadotrophinsb
Days of ovarian stimulationb
Patients with HCG and ovum retrieval (n,%)
No of >10 mm follicles on HCG day*
No. of »14 mm follicles on HCG day6
Endometrial thickness (mm) on HCG day*1
Oestradiol (pg/ml)c on HCG dayb
Study 2
pFSH (n = 92)
HMG-1 (n = 96)
FSH-HP (n = 123)
HMG-2 (n = 129)
13 7 ± 2.1
30 0 ± 5 . 8
10 2 ± 2.3
73 (79)
15 8 ± 72
128 ± 7 0
11.5 ± 2.7
2397 ± 1571
14.0 ± 2 6
29.5 ± 5 1
10.1 ± 2 1
74 (77)
15.1 ± 6 4
119 ± 5.5
11.0 ± 2.3
2572 ± 2065
138 ± 14
29 2 ± 6.1
9.8 ± 1.4
94(76)
154 ± 6 4
12 3 ± 6 5
10.7 ± 2.1
2094 ± 1627
14 1 ± 1 2
29 2 ± 5 8
10 1 ± 1 7
105 (81)
14.5 ± 6 0
11 1 ± 5 5
10.5 ± 2 1
2407 ± 1848
'No significant differences between pFSH and HMG-1 groups nor between FSH-HP and HMG-2 groups.
•"Values are means ± SD.
Conversion factor to SI unit, 3.671
HCG = human chononic gonadotrophin, FSH = follicle stimulating hormone, pFSH = pure FSH, FSH-HP = highly purified FSH, HMG = human
menopausal gonadotrophin.
Table i n . Ovum retrieval and IVF outcome in the four groups studied
Study 2
Study 1
Variable'
pFSH (n = 92)
HMG-1 (n = 96)
FSH-HP (n - 123)
HMG-2 (n - 129)
No. of follicles puncturedb
No. of oocytesb
Mature oocytes (%)
Immature oocytes (%)
Degenerative oocytes (%)
No. of fertilized oocytesb
No. of embryos/patientb
No. of embryos/replacementb
Patients with embryo transfer (n,%)c
Clinical pregnancy rates (n)
Cycle start (%)
Ovum retrieval (%)
Embryo transfer (%)
Clinical abortion rates (n,%)
19.4 ± 11.7
95 ± 58
95
3
2
72 ± 50
6.6 ± 5 0
2.8 ± 0 7
62 (84.9)
13
14 1
17.8
20.9
2 (15.3)
18.2 ± 10.5
9.3 ± 6 4
93
4
3
6.2 ± 5 6
5.7 ± 5 0
2 6 ± 0.5
63 (85 1)
11
114
14.8
17.4
2(18 1)
19 1 ± 11.2
9 3 ± 5.6
96
3
1
6 1 ± 5.0
5 6 ± 4.5
28 ± 0 4
85 (90.4)
16
13.0
17 0
18.8
2 (12.5)
17 7 ± 8.3
96 ± 49
98
1
1
66 ± 45
58 ± 42
29 ± 06
98 (93 3)
21
16 2
20.0
214
4(19 0)
•No significant differences berween pFSH and HMG-1 groups nor between FSH-HP and HMG-2 groups
"Values are means ± SD
°Values are relative to the number of patients with ovum retrieval.
similar in the four groups of patients (Table HI). There
were no ectopic pregnancies. The overall incidence of severe
ovarian hyperstimulation syndrome (Golan et al., 1989) was
1.8% (eight cases among 440 IVF cycles) and there was
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one case (1%) in group pFSH, two cases (2%) in group
HMG-1, two cases (1.6%) in group FSH-HP and three cases
(2.3%) in group HMG-2 (no statistical significant difference
between groups).
FSH versus FSH/HMG after GnRHa
Discussion
The most exciting development of ovarian stimulation for IVF
was the widespread use of the GnRHa during therapy with
HMG. This prevents inappropriate release of a spontaneous
LH surge and allows optimal timing of oocyte collection to
an ovulation-inducing injection of HCG (Hughes et al, 1992;
Meldrum, 1993; Tan et al, 1994). Recent studies suggested
that the choice of GnRHa may influence results obtained in
IVF (Balasch et al, 1992; Penzias et al, 1992). Similarly, it
has been stressed that the traditional administration of equal
amounts of urinary FSH and LH needs to be reviewed m the
light of advancing knowledge of basic follicular physiology,
pharmaceutical developments in the specificity and purity of
both FSH and LH preparations, and growing clinical experience
with such preparations (Hillier, 1994; Hull et al, 1994).
The availability of pFSH and FSH-HP made it possible to
discriminate better the relative roles of FSH and LH in
the ovulation process, underlining the key role of FSH in
folliculogenesis and has afforded a greater flexibility in terms
of gonadotrophin utilization.
Whereas hypersecretion of endogenous LH during the follicular phase may imply adverse effects on the fertility process
(Shoham et al, 1993), exogenous LH seems to have very little
impact on endogenous LH both in polycystic ovary syndrome
patients undergoing ovulation induction (Hamilton-Fairley
et al., 1991; Sagle et al, 1991) and in eumenorrhoeic women
given gonadotrophin and long regimen GnRHa for ovarian
stimulation in IVF (Filicori et al, 1996). However, the long
protocol of GnRHa administration in combination with the
use of pure preparations of FSH is now being increasingly used.
Recently, two non-comparative studies have been reported
indicating that both pFSH (Hull et al, 1994) and FSH-HP
(Howies et al, 1994), when used to stimulate ovaries in IVF
cycles employing pituitary desensitization, are fully effective
and highly successful, and supplementation with LH is not
needed. On the contrary, two very recent reports indicate that
just replacing HMG with FSH-HP may not be good enough
for ovarian stimulation of GnRHa down-regulated, normogonadotrophic women (Fried et al, 1996; Westergaard et al, 1996).
Serum oestradiol concentrations were lower in FSH-HP groups
and some beneficial effects of HMG on the average number
of oocytes retrieved/fertilzed per cycle and pre-embryo development were found in those studies.
To the best of our knowledge, no study has been reported
comparing FSH-HP alone with the combination FSH-HP/
HMG in IVF. The present report shows that there is no
significant difference between these two gonadotrophin regimens of ovarian stimulation when used in conjunction with
GnRHa. This is indicated by the similarity in overall follicular
growth, oocyte recovery and IVF outcome. Similarly, we found
that patients treated with GnRHa had identical stimulation,
oocyte retrieval and pregnancy outcomes, if they were stimulated with pFSH alone or pFSH/HMG. Although mean serum
oestradiol concentrations tended to be slightly higher in the
combination treatment groups, differences were not statistically
significant This is in agreement with recent studies showing
that whereas in extreme hypogonadotrophic women LH con-
centrations are below the LH threshold for normal oestradiol
biosynthesis, full follicular maturity and oocyte fertility, this
threshold is not surpassed by GnRHa down-regulation in IVF
(Hillier, 1994; Balasch et al, 1995; Simoni and Nieschlag,
1995). The endocrine role of oestradiol on endometnum was
adequate in the four treatment groups as supported by the
similarity observed in the endometrial thickness on HCG day
and the pregnancy rate and outcome. On the other hand, while
the 1.8% overall incidence of severe ovarian hyperstimulation
syndrome in our study was very similar to the 1.7% recently
reported by Dahl Lyons et al. (1994) among a similar number
of IVF cycles stimulated according to a protocol such as ours
(long protocol of GnRHa plus gonadotrophins), we found no
difference between groups with respect to the incidence of
this complication.
A recent meta-analysis including eight randomized trials of
pFSH versus HMG use in ovarian stimulation suggests that
the use of pFSH is associated with a significantly higher
clinical pregnancy rate (Daya et al, 1995). However, while
the overall OR m that meta-analysis was in favour of the use
of pFSH, in each trial (including the one by the authors of
meta-analysis) the OR was not statistically significant. It should
be noted that seven of these eight studies analysed only
included about 20 patients in each treatment group (Daya
et al, 1995). In addition, the meta-analysis included trials both
with and without GnRHa, and trials were also included whether
the ovarian stimulation regimen included GnRHa using the
short flare-up protocol, in which GnRHa was commenced in die
follicular phase of the treatment cycle, or the long suppression
protocol, in which GnRHa was commenced in the follicular
phase of the treatment cycle or the luteal phase of die preceding
cycle (Daya et al., 1995). This suggests that endogenous LH
concentrations were markedly different from one study to
anouier. Thus, the small number of patients included and
marked differences in GnRHa regimen used in those previous
studies make it difficult to obtain definite conclusions regarding
the optimum gonadotrophin regimen for IVF. In our study
no differences were found between pFSH and pFSH/HMG
treated groups.
In conclusion, we have been unable to find significant
differences in follicular development, the number of ova and
embryos, or pregnancy rates between patients stimulated with
conventional urinary FSH/HMG dierapy and those given
urinary FSH alone under pituitary suppression with GnRHa.
Thus, both gonadotrophin regimens can be used indistinguishably. Further studies are necessary to improve the stimulation
protocols in order to obtain die optimum hormonal milieu for
maximum follicular development and oocyte harvest as well
as the ideal milieu for initiating and maintaining implantation
in IVF-embryo transfer.
Acknowledgement
The authors thank Mrs Paquita Antonell for her technical assistance.
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Received on June 13, 1996; accepted on September 5, 1996
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