RBMOnline - Vol 5. Suppl. 1. 73–86 Reproductive BioMedicine Online; www.rbmonline.

com/Article/697 on web 13 September 2002

Ovarian stimulation: from basic science to

clinical application
Michael Ludwig1,3, Ricardo E Felberbaum1, Klaus Diedrich1, Bruno Lunenfeld2
1Division of Reproductive Medicine and Gynecologic Endocrinology, Department of Gynecology and Obstetrics,
University Clinic, Ratzeburger Allee 160, 23538 Lübeck, Germany
2Faculty of Life Sciences, Bar–Ilan University, Ramat Gan, 52900 Israel
3Correspondence: Tel: +49 451 5002158; Fax: +49 451 5059334; e-mail: Ludwig_M@t-online.de

Abstract
Treatment for infertility, including ovarian stimulation, was first introduced almost 100 years ago. At this time, radiation
therapy became an established treatment, and it was only some decades later that the problem of radiation-induced cancer
emerged. Non-human gonadotrophins, such as pregnant mare serum gonadotrophin (PMSG), and human pituitary
gonadotrophins (HPG), were commonly used for hormonal stimulation procedures. However, use of PMSG led to antibody
formation, and it was therefore only useful for the first treatment cycle. HPG produced good results, but its use came to an
end in the late 1980s when it was linked to the development of Creutzfeldt–Jakob disease. The first hormonal product from
human menopausal urine to be used was human menopausal gonadotrophin (HMG), followed later by purified preparations
of this product. All of these preparations contained a high percentage of unknown urinary proteins, which interfered with
batch-to-batch consistency. This changed with the introduction of recombinant gonadotrophins, produced from an
immortalized/standardized mammalian cell line (CHO). More recent developments include the introduction of long-acting
gonadotrophin formulations. The development of gonadotrophin-releasing hormone (GnRH) analogues and more recently
the use of GnRH antagonists has helped to improve ovarian stimulation protocols by optimizing their efficacy, and making
them easier to administer.

Keywords: FSH, GnRH antagonist, gonadotrophin formulation, LH, recombinant gonadotrophin, urinary gonadotrophin

Introduction
Infertility is not an entity of our century – it is as old as
mankind. The Bible tells the story of several infertile women
(Figure 1) (Lunenfeld, 1995). At that time, however, fertility
was believed to be a gift from God, which could be given and
taken away, depending on what people did or did not do.
Nowadays there is an ongoing discussion as to whether the
prevalence of infertility is increasing. Statistics from around
1900, from Australia, have estimated the prevalence of
infertility in married couples to be 11% (Cummins, 1999), a
similar figure to that observed today (Snick et al., 1997).
As knowledge on the aetiology of infertility has increased over
the years, treatment options have changed, and a number of
different assisted reproductive techniques are now available.
The success of these techniques depends on many factors,
including the age of the patient, the patient’s medical history,
and the ovarian stimulation regimen used. For IVF and
intracytoplasmic sperm injection (ICSI), laboratory
conditions, the number of embryos replaced and the skill of the
embryologist also have to be considered.
The history of the use of gonadotrophins in the treatment of
infertility extends from early attempts at their extraction from
animals, human cadavers and human urine, to the production
of recombinant products from CHO cells. During the last 15
years, the development of gonadotrophin-releasing hormone
(GnRH) analogues has helped to improve ovarian stimulation
protocols by optimizing their efficacy, and making them easier
for doctors to administer and more comfortable for patients to
receive. All along, the development of gonadotrophins for use
in infertile women has been driven by the need to make these
products effective, safe, pure and consistent, thereby reducing
treatment variability.
Some of the leading scientists and clinicians who have
contributed to this history over the past 100 years are shown in
Figure 2.
Ovarian and pituitary irradiation for
the treatment of infertility
In the early 1900s, it was proposed that irradiation of the
ovaries might be a useful treatment for infertility by
stimulating ovarian function (Halberstaedter, 1905). In
addition, beneficial effects of Roentgen irradiation of the
pituitary were reported (Beclere, 1926). As a result, the
techniques of ovarian and pituitary irradiation became widely
established as treatments for infertile women in the USA
(Rubin, 1926; Mazer and Greenberg, 1943; Kaplan, 1948).
This resulted in a statement by Rita Finkler, in 1949, that:
‘Irradiations of the ovarian and pituitary regions for the relief
of functional sterility, in the female is a universally accepted
therapeutic procedure’ (Finkler, 1949).
The benefits of radiation therapy compared with endocrine
therapy were demonstrated in a study of 130 patients who
underwent irradiation of the ovaries and pituitary at a dose of
200 ~kV. Such treatment was reported to require shorter
treatment regimens, to be less expensive, and to be easier to
administer than endocrine therapy (Finkler, 1949). The
cumulative pregnancy rates after a 2-week course of radiation
therapy are shown in Figure 3. It was concluded that the 73

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 Ovarian stimulation: from basic science to clinical application - M Ludwig et al.

Figure 1. Infertility in the bible. Abraham was married to

Sarah. Sarah, however, was unable to become pregnant, and
therefore she gave her maid Hagar to Abraham instead;
perhaps the first surrogate mother. Hagar became pregnant
with Ishmael. Sarah subsequently became pregnant herself –
as written in the Bible, due to the will of God – and gave birth
to Isaac. Isaac, after other wives, married Rebecca, who
initially was infertile. Only after Isaac had asked God for
fertility in his marriage, did God take away her infertility from
Rebecca and she gave birth to twins, Esau and Jacob. Rachel,
Jacob’s wife, was infertile, too, due to the will of God, since
God wanted to punish Jacob, who loved Rachel more than
Leah, his other wife. Finally, however, after Leah had given
birth to several sons, God made Rachel fertile and she gave
birth to Joseph. Before that, due to her infertility, she, just like
Sarah, gave her maid Bilha to Jacob and Bilha gave birth to Figure 2. Some of the pioneers who pushed the development
two sons (Genesis 16–30) (adapted from Lunenfeld, 1995). of gonadotrophins forward, either by clarifying the
physiological background or by the development of innovative
preparations. Selmar Aschheim (a), Bernhard Aschner (b),
Gerhard Bettendorf (c), Pietro Donini (d), Bruno Lunenfeld
cumulative conception rates were independent of the treatment
(e), Carl Axel Gemzell (f), Philip Edward Smith (g) and
administered, being 34.2% among patients receiving hormonal
Bernhard Zondek (h). All of the pictures, with the exception of
therapy and 35.2% among those receiving radiation therapy. In
(e), were reprinted with kind permission of Springer Verlag
patients suffering from secondary amenorrhoea, menstrual
[from: G. Bettendorf (1995) Zur Geschichte der
bleeding was re-established in 40.6% of those receiving
Endokrinologie und Reproduktionsmedizin. Springer Verlag,
hormonal therapy and 46.4% of those receiving radiation
pp. 12, 15, 37, 117, 174, 539, 632].
therapy (Finkler, 1949). However, we now know that the
stimulatory effect of irradiation may have been due to
increased blood flow and hyperaemia. The price of radiation
therapy did not emerge until some 45 years later, around 1985, function of higher centres in the brain. This hypothesis was
when the National Institute of Health (NIH) and others based on his observations in men and women with head trauma
reported that many patients who had received this kind of that lesions between the hypothalamus and the pituitary lead to
therapy had developed ovarian cancer (Ron et al., 1994). hypopituitarism and gonadal atrophy (Aschner, 1912).
Therefore, he was the first to propose a hypothalamic–pituitary
Early understanding of the interaction for gonadotrophic action.
hypothalamic–pituitary–ovarian axis In 1926, Smith and Engle demonstrated that immature male or
The pituitary was already known in Ancient Greece. They female animals that were hypophysectomized failed to mature
called it hypophysis, or ‘something which grows at the bottom sexually. They also showed that daily transplants of anterior
of the brain’. Later on, the physician Galen proposed, that the pituitary gland tissue from mice, rats, cats, rabbits and guinea
hypophysis is a kind of trashcan, where metabolites from the pigs into immature male and female mice and rats rapidly
brain are collected. He therefore called this structure glandula induced precocious sexual maturity and superovulation
pituitaria, because he thought that the trash was brought to the (Smith, 1926; Smith and Engle, 1927). In the same year, the
nose as pituita, nose mucus. Only in the seventeenth century pioneering experiments by Zondek showed that ovarian
did it became clear that under normal conditions, there is no function is regulated by the pituitary: the implantation of
flow of any fluid from the brain to the nose, and that the anterior pituitary glands of adult cows, bulls and humans into
pituitary must have another function. immature animals was shown to evoke the rapid development
of sexual puberty (Zondek, 1926).
Work by Crowe et al. in 1910 showed that partial ablation of
the pituitary gland leads to atrophy of the genital organs in It was again Zondek, only 4 years later, who proposed the
adult dogs, and to the persistence of infantilism and sexual secretion of two hormones by the pituitary:
inadequacy in puppies (Crowe et al., 1910). Two years later, ‘Follikelreifungshormon’ or Prolan A, and
74 Aschner postulated that pituitary function depends on the ‘Luteinisierungshormon’ or Prolan B (Zondek, 1930) (Figure

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 Ovarian stimulation: from basic science to clinical application - M Ludwig et al.

Cumulative pregnancy rate

Months
Figure 3. Effect of a 2-week course of radiation therapy on ovaries and the pituitary in cases of ‘functional infertility’, i.e.
infertility due to abnormal hormonal values or abnormal bleeding patterns without a male factor. Eighty rads was delivered into
each ovary and into the pituitary gland. Shown are cumulative pregnancy rates in a group of 54 patients. Seventeen of these
patients were ‘endocrine resistant’, i.e. they did not get pregnant after combined PMSG/HCG treatment. In another series, the
authors showed a cumulative pregnancy rate of 47%; in 28 amenorrhoeic patients they were able to re-establish menses in 46.4%
(Finkler, 1949).

4). This hypothesis was proved only a year later with the
extraction of two different hormones from the pituitary, one of
which acted as a follicle stimulating factor and one as a
luteinizing factor (Fevold et al., 1931).

The third gonadotrophin, human chorionic gonadotrophin

(HCG), was first described in 1943, when it was shown to be
secreted from trophoblastic cells. Zondek and Aschheim, after
administration of urine from pregnant individuals, showed the
development of follicular cysts and hemorrhagic follicles in
the mouse – the first pregnancy test (Zondek and Aschheim,
1927). Such changes are nowadays commonly seen in ovarian
hyperstimulation syndrome (OHSS).

The link between the hypothalamus and the pituitary was

finally proposed by Guillemin in 1967, when he suggested that
an LH-releasing factor, now termed GnRH, controlled the
release of gonadotrophins from the pituitary for follicular
maturation (Guillemin, 1967). The race between two different
laboratories to elucidate the structure of this GnRH finally
ended in June 1971, with a report by Andrew Schally and
colleagues at the annual meeting of the Endocrine Society in
San Francisco of the decapeptide structure of GnRH (Schally
et al., 1971). The group had analysed 240 μg of the compound,
amalgamated from 160,000 pig pituitaries.

Figure 4. The original figure (Zondek, 1930) showing the
proposed connection between pituitary and ovaries, as well as
the action of Prolan A (‘Follikelreifungshormon’) and Prolan B
(‘Luteinisierungshormon’). The graph shows the action of
Prolan A on the secondary follicle, resulting in maturation to
the Graafian follicle. After ‘Follikelsprung’ (ovulation), a
corpus luteum results, which, following the idea of Professor
Zondek, is under the influence of Prolan B. Furthermore, the
action of follicular hormones on the endometrium is shown in
its different phases (‘Proliferationsphase’ and
‘Sekretionsphase’). Figure reprinted with kind permission of
Springer Verlag.
The next steps in the learning process were the discoveries that
the GnRH gene is located on chromosome 8, and that GnRH is
produced from a pre-hormone known as Pre-Pro-GnRH
(Wetsel et al., 1991, 1995). GnRH was also shown to be
rapidly metabolized, with a half-life of <5 min, and to be
mediated by a transmembrane receptor, which stimulates the
release of LH and FSH. This receptor was shown to be a
calcium-mobilizing, seven transmembrane domain receptor
that is coupled to phospholipase Cβ though G proteins (Yen,
1999).
It was Knobil who contributed with his pioneering work to the
discovery of the interaction between the hypothalamus and
pituitary by GnRH. Circhoral LH pulses were assumed to be
the consequence of pulsatile GnRH secretion by the
hypothalamus. This gave rise to the idea, that an oscillator or
signal generator in the central nervous system, the so-called
GnRH pulse generator, must exist (Knobil, 1974, 1980). He
also showed, that continuous administration of GnRH results 75

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 Ovarian stimulation: from basic science to clinical application - M Ludwig et al.
b
in pituitary suppression. Pulsatile GnRH administration was
necessary to produce an adequate pituitary response and LH
secretion (Knobil, 1980, 1988; Wildt et al., 1986). Thus,
GnRH can either stimulate or inhibit gonadotrophin secretion,
depending on its mode of administration (Plant et al., 1978;
Wildt et al., 1981).
Introduction of pregnant mare
serum gonadotrophin (PMSG)
In 1930, Cole and Hart showed that pregnant mare serum
gonadotrophin (PMSG), produced in the endometrial cups of
pregnant mares, has potent gonadotrophic activity (Cole and
Hart, 1930). Thus in 1937, PMSG was extracted for use in
anovulatory women. However, this extract produced
inconsistent results and adverse effects. In 1941, the concept of
the two-step protocol was introduced: ovarian stimulation
using gonadotrophins and the induction of ovulation using
HCG. Mazer and Ravetz used PMSG in this protocol in
amenorrhoeic women with good results: 23 severely
amenorrhoeic women received either PMSG or a pituitary
extract from pigs and sheep for follicle stimulation, and HCG
for the induction of ovulation. Menstruation was induced in 19
of the patients, some of whom had not menstruated for years.
These results suggested the presence of a pituitary factor that
stimulated mature follicle development (Mazer, 1946).
In 1942, however, a number of research groups described the
development of ‘antihormones’ in women who were treated
76 with PMSG. This was essentially an antigen–antibody reaction.
S6
Figure 5. Production of urinary gonadotrophins. Shown is
the collection of pooled urine from postmenopausal
women (a), the collection in large tanks (b), and the
manual processing of a Kaolin filter after centrifugation
(c). All pictures reprinted with kind permission of Serono
International S.A., Geneva, Switzerland.
Patients who did not get pregnant after the first or second
treatment cycle using PMSG became insensitive to it, and this
marked the end of the PMSG era (Ostergaard, 1942; Zondek and
Sulman, 1942). Interestingly, PMSG was still used in Eastern
Germany until 1973 (Groot-Wassink and Blawert, 1973).
Human pituitary gonadotrophins
(HPG)
In, 1958, another source of gonadotrophins was discovered:
the pituitary glands from human cadavers. The gonadotrophins
extracted from this source were termed human pituitary
gonadotrophin (HPG) (Gemzell et al., 1958; Bettendorf,
1963). HPG was shown to induce ovarian stimulation in
hypophysectomized individuals (Bettendorf, 1963), and was
used extensively for the induction of ovulation until 1988.
At this time, HPG was withdrawn from the market because of
the discovery of 12 cases of Creutzfeldt–Jakob disease that
were thought to be linked to the use of this product (Dumble
and Klein, 1992). Interestingly, however, these cases of
Creutzfeldt–Jakob disease did not occur in patients who
received pharmaceutically produced products but in patients
who received government-produced products: from the
Pituitary Agency in Australia, the Pituitary Agency in the UK,
and the Pituitary Agency of France. HPG was withdrawn from
the market after that, and another period of gonadotrophins
came to its end (Cochius et al., 1990).
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 Ovarian stimulation: from basic science to clinical application - M Ludwig et al.

Figure 6. Purification of human postmenopausal urine to retrieve HMG. First, the urine is passed through a column containing
kaolin. This produces a solution containing urinary FSH, LH and 95% urinary proteins (a). Polyvalent antibodies to HCG are then
bound into the kaolin column, which holds back the urinary LH but allows urinary FSH to pass through. The result is a mixture
of urinary FSH and 95% urinary proteins (b). The final step involves the introduction of a monoclonal anti-FSH antibody into a
Sepharose column, followed by the elution of bound FSH from the column (c).

basis on the future of gonadotrophins, as well as on the

Human menopausal gonadotrophin
(HMG)
Human menopausal gonadotrophin (HMG) was developed at
around the same time as HPG. This gonadotrophin is present
in the urine of postmenopausal women (Figure 5), and was
extracted using a technique developed by a Serono chemist in
1949 (Donini et al., 1964), in which kaolin is used to absorb
glycoproteins from the urine (Figure 6). The gonadotrophin
thus produced was shown to have about 5% biological activity
and to consist of 95% urinary proteins. Administration of this
gonadotrophin in an animal model resulted in an increase in
the size of the ovaries and uteri, therefore proving its
effectiveness in ovarian stimulation (Borth et al., 1954).
In 1953, HMG was successfully used to stimulate the ovaries
of hypophysectomized rats (Borth et al., 1954), and in 1959,
its use in humans was shown to cause dramatic changes in
steroid excretion, and in the endometrium and vaginal
epithelium (Lunenfeld et al., 1960). The first live births
following the administration of HMG to humans were reported
in 1962 (Lunenfeld et al., 1962).
At around this time, the threshold hypothesis was developed:
this suggested that an enlargement of the FSH window would
lead to an increase in the number of developing follicles in the
early follicular phase (Lunenfeld et al., 1961). This hypothesis
was picked up in later years and elegantly described as
’threshold therapy’. The hypothesis also led to the development
of different ovarian stimulation procedures (Figure 7).
standardization of these new products. In the summer of 1953,
several scientists met in Geneva, Switzerland, to define basic
and clinical goals for gonadotrophic research. Some of the
members of this first meeting of the ‘Gonadotrophin Club’ (G-
Club) were Rudi Borth, Bruno Lunenfeld, Hubert de
Watteville, Egon Diczfalusy, Jim Brown, John Loraine, and
A.C. Crooke. The participants agreed to develop specific assay
procedures, bioassay standards and purification methods, that
would lead to the development of a gonadotrophic preparation
for therapeutic purposes. It was also their work to first define
the term ‘human menopausal gonadotrophin’ (HMG) for the
newly made preparation. The second meeting of the G-Club
was held in Birmingham, where the main topic was the
chemical and biological properties of gonadotrophins
extracted from human fluids.
In 1959 another G-Club meeting led to the agreement that a
reference batch of gonadotrophin was needed, to standardize
treatment with gonadotrophins. In 1960, this resulted in the
decision that a batch of 50 g Pergonal® (human menopausal
gonadotrophin; Serono International S.A.), lot 23, should be
the reference preparation. Four years of discussion followed
and an international collaborative study was organized to
calibrate the FSH and LH activities in this batch. Eventually,
in 1964, the Second International Reference Preparation-HMG
was established.

As a result of these rapid developments in this field, in the

mid-1950s the G-Club formed, to discuss on an international 77

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 Ovarian stimulation: from basic science to clinical application - M Ludwig et al.

Figure 7. Step-up (a), step-down (b) and sequential protocol

(c) (Lunenfeld, 1963; Fauser et al., 1993; Hugues et al., 1996).
Whilst the step-up protocol (a) seems to be ideal for patients
suffering from polycystic ovarian syndrome, since it helps to
avoid multifollicular development, others have described, for
the same cohort of patients, the step-down protocol (b) as the
ideal approach. Each of the three protocols has been proven to
c work and may have its benefits as well as drawbacks.

Table 1. WHO classification scheme for cases of ovarian

Ovarian hyperstimulation hyperstimulation syndrome (OHSS) (World Health
syndrome: the problem of Organization, 1973). This historical scheme has been
gonadotrophin administration modified several times, but it remains the one used in most
studies for classification of this entity.
Soon after HMG preparations were first used in ovarian
stimulation procedures, the first cases of OHSS were reported Grade Findings Treatment
in the literature (Mozes et al., 1965). As a result, the World
Health Organisation proposed a classification and I Variable ovarian No treatment
management scheme for cases of OHSS (Table 1) (Melmed et enlargement necessary
al., 1969). Even now, OHSS is the most important Sometimes ovarian Report additional
complication of ovarian stimulation. It can itself lead to further cysts symptoms to the physician
complications (Ludwig et al., 1999b, 2000), and may be life Increased urine
threatening (Cluroe and Synek, 1995). hormones
II Abdominal Careful medical
LH and FSH in ovarian stimulation distension observation
protocols: the two-cell theory Nausea Symptomatic treatment
Vomiting
The two-cell two-gonadotrophin theory was proposed in 1966 Diarrhoea
(Ryan and Petro, 1966; Ryan, 1979). Essentially, this states III Large ovarian Hospitalization and
that LH binds to receptors on the thecal cells, stimulating the cysts prompt medical treatment
production of androgens. These diffuse to the granulosa cells, Ascites Correction of altered fluid
where they are then transformed into oestrogens by the and electrolyte balance
aromatase produced by the granulosa cells, stimulated by FSH. Sometimes
hydrothorax
With respect to LH, it is now well known that there is a certain Haemoconcentration
threshold concentration of this gonadotrophin below which may appear
78 oocyte maturation will not occur. This situation is observed in

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 Ovarian stimulation: from basic science to clinical application - M Ludwig et al.
Table 2. Results of a meta-analysis of eight prospective, randomized trials comparing urinary FSH and
HMG in IVF treatments. Included were ovarian stimulation protocols with and without the use of GnRH
agonists. Data according to Daya et al. (1995).
Analysis per Gonadotrophin Clinical
used pregnancy (%)
Cycle start FSH 63/285 (22.1)
HMG 45/332 (13.6)
Oocyte retrieval FSH 63/268 (23.5)
HMG 45/291 (15.5)
Embryo transfer FSH 63/254 (24.8)
HMG 45/273 (16.5)
women with WHO I ovarian insufficiency, such patients
showing little or no gonadotrophic activity. In these patients,
oestrogen production and endometrial growth occur only with
the administration of exogenous LH, as demonstrated in a
prospective randomized study (The European Recombinant
Human LH Study Group, (1998).
However, there is now overwhelming evidence to show that
inappropriately high concentrations of LH also have a
detrimental effect on fertility: high concentrations of LH
appear to have a direct effect on the follicles, leading to
premature luteinization, follicular atresia and poor-quality
oocytes. These, in turn, result in higher abortion and lower
ongoing pregnancy rates (Stanger and Yovich, 1985; Ludwig
et al., 1999a).
Purification of HMG
As long ago as 1973, the World Health Organization (WHO)
(Figure 8) stated that: ‘The ratio of FSH to LH varies in
different HMG and HPG preparations, but the available
evidence indicates, that preparations with ratios of 0.1–10.0
are acceptable therapeutic agents provided, that a sufficient
total dosage of FSH is administered to the patient’ (WHO,
1973). A preparation with such high variation (Rodgers et al.,
1994) is not ideal for clinical use. Furthermore, HCG is
sometimes added to HMG in order to increase the LH content
and achieve the labelled activity of 75 IU per ampoule. HCG
has been detected in all available HMG products, irrespective
of their declared purity (Stokman et al., 1993). Since the half-
life of HCG is significantly longer than that of LH, the variable
amounts of HCG added may contribute to the batch-to-batch
variations.
In view of the detrimental effect of high LH concentrations on
fertility, attempts were made to minimize LH activity in
preparations of HMG. This was achieved by extracting the LH
using a polyvalent antibody to HCG in a kaolin column,
leaving a solution containing only FSH and urinary proteins
(Donini et al., 1966; Eshkol and Lunenfeld, 1967). Serono
Laboratories further refined this purification technique by
introducing monoclonal anti-FSH antibodies into the kaolin
column and eluting FSH from the column by breaking the
antigen–antibody complex. This process increased the FSH
concentration in the gonadotrophin preparation from 100 to
10,000 IU/mg protein (urinary FSH highly purified [uFSH-
Reprinted from Vol. 5, Suppl. 1 (2002) pp. 73 86.
Absolute Odds ratio P-value
treatment effect (95% CI)
in favour of
FSH (%)
8.5 1.71 (1.12–2.62) 0.009
8.0 1.69 (1.10–2.59) 0.016
8.3 1.70 (1.10–2.62) 0.018
HP]; Metrodin HP®, Serono International S.A., Geneva,
Switzerland) (le Cotonnec et al., 1993; Howles et al., 1994).
Several prospective, randomized studies were then performed
to determine the efficacy of highly purified FSH for ovarian
stimulation. A meta-analysis of eight of these studies clearly
showed that purified FSH was more effective in this respect
than HMG, increasing the pregnancy rate by approximately
1.7 fold (Table 2) (Daya et al., 1995).The use of FSH either
alone or following contraceptive pills and/or in combination
with GnRH analogues became the gold standard for patients
with PCOD receiving assisted reproductive treatment.
However, a major problem with the production of highly
purified FSH was the need for large quantities of urine from
postmenopausal women. When production of urinary
gonadotrophins began, there were three urine collecting
centres, with about 600 donors who produced 120,000 l of
urine annually: one in the Netherlands, one in Spain, and one
in Italy. At the start of the 1990s it was estimated that 120 ×
106 million litres of urine would be needed to satisfy the
worldwide demand for pure FSH, and this would require the
services of about 600,000 donors. Clearly, a different source of
pure FSH was needed (Rodgers et al., 1994; Giudice et al.,
2001).
This increased need was due to the increasing number of
indications. In the early 1960s HMG was used as replacement
therapy in WHO I patients, in the late 1960s, HMG was used
as regulation therapy in WHO II patients who did not respond
or who did not conceive following three cycles with
clomiphene citrate, from the mid-1970s it was increasingly
used for ovarian stimulation for IVF in infertile patients with
tubal pathology, and in the 1990s there was an exponential
increase when it was used for ICSI in cases of severe male
factor infertility. In fact, the first IVF pregnancy, in 1975, was
the result of HMG stimulation, although it ended as an ectopic
pregnancy (Steptoe and Edwards, 1976). Louise Brown was
then born following natural cycle IVF in 1978 (Steptoe and
Edwards, 1978). By 2–3 years later, babies had been born in
Australia and France, then in USA in 1982.
Development of recombinant
gonadotrophins
The production of gonadotrophins by recombinant genetic
engineering technology was finally made possible by the
79
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 Ovarian stimulation: from basic science to clinical application - M Ludwig et al.
Figure 8. The title page of the WHO paper on ‘Agents
stimulating gonadal function in the human’. The members of
the scientific board (reproduced with kind permission of the
WHO).
isolation of the human genes coding for the common alpha
subunit and the specific beta subunits. This led to the
preparation of appropriate vectors, which were transfected into
a suitable immortalized mammalian cell line namely, the
Chinese hamster ovary (CHO) cell line. A cell line
characterized by a high and stable level of FSH expression was
isolated from the transfected cells: CHO DUKI with two
plasmids (follitropin alpha; Gonal F®, Serono International
S.A., Geneva, Switzerland) and CHO KI with one plasmid
(follitropin beta; Puregon®, Organon, Oss, The Netherlands).
These cell lines were used to establish a master cell bank,
which now serves as the source of working cell banks (WCB).
Thus, a continuous supply of recombinant human FSH with
guaranteed consistency from WCB to WCB is now available
(Howles, 1996). A modern bioreactor used in the manufacture
of recombinant human FSH (follitropin alpha) is shown in
Figure 9.
The advantages of using recombinant human FSH include the
availability of unlimited quantities of the hormone, guaranteed
quality of the source of the product and the final product,
batch-to-batch consistency, and the availability of a highly
pure preparation that is not contaminated with unknown
urinary proteins (Hugues, 2001; Risquez, 2001). Furthermore,
a recent meta-analysis has shown that recombinant human
FSH is superior to urinary FSH (Table 3) (Daya, 2002).
Recent cost-effectiveness analyses have also shown that the
higher costs of recombinant human FSH compared with
urinary FSH are outweighed by the higher success rates
80 achieved with the former preparation. Even if all these studies
S10
Figure 9. A modern bioreactor for the production of follitropin
alpha (Gonal F®). Picture reprinted with kind permission of
Serono International S.A., Geneva, Switzerland.
are using mathematical statistical models for the calculation of
costs, they are a helpful tool under these circumstances.
In one UK study, a decision-analytic model was used to
compare the results achieved with the recombinant human
FSH follitropin beta and those achieved with urinary FSH and
HMG. The cost of one IVF cycle was £5135 for recombinant
human FSH, £4806 for urinary FSH, and £4202 for HMG. For
one ongoing pregnancy, the costs were in favour of
recombinant human FSH (£8992 versus £10,834 for urinary
FSH and £9472 for HMG (Sykes et al., 2001). Another UK
study used a Markovian decision framework and Monte Carlo
simulation to compare the costs of treatment with the
recombinant human FSH follitropin alpha and urinary FSH. In
this study, the cost per successful pregnancy was significantly
lower for recombinant human FSH (£5906) than for urinary
FSH (£6060) (Daya et al., 2001). Similarly, a Markov model
analysis performed in the United States gave costs per
successful pregnancy of $40,688 for recombinant human FSH
(follitropin alpha) and $47,096 for urinary FSH (Silverberg et
al., 2002a,b).
Following the successful production of recombinant human
FSH, similar technology was used to produce other
Reprinted from Vol. 5, Suppl. 1 (2002) pp. 73 86.
 Ovarian stimulation: from basic science to clinical application - M Ludwig et al.

Table 3. Results of a meta-analysis comparing recombinant FSH and urinary FSH in

ovarian stimulation for IVF cycles (data according to Daya, 2002). Follitropin alpha:
Gonal F®, Follitropin beta: Puregon®.

Type of procedure and Common odds ratio Risk difference (%)

follitropin used (95% confidence interval) (95% confidence interval)

IVF
Follitropin alpha 1.37 (1.05–1.79) 5.4 (0.8–10.1)
Follitropin beta 1.19 (0.95–1.49) 3.5 (–1.0–7.9)
ICSI
Follitropin alpha 1.04 (0.73–1.47) 0.8 (–6.7–8.3)
Follitropin beta No data available No data available
IVF or ICSI
Overall total 1.21 (1.04–1.42)

Table 4. Milestones in the development of recombinant human gonadotrophins (data modified

according to Howles, 1996).

1972 First recombinant DNA molecules generated at Stanford University, CA, USA
1979 Human insulin was cloned in bacterial cells
1979 Human chorionic gonadotrophin α-subunit cloned
1983 Amino acid sequence of human β-FSH subunit established
1985 Human β-FSH gene cloned, and biologically active FSH was expressed in
mouse fibroblast cells
1988 Human FSH expressed in continuous mammalian cell line (CHO)
1989–1990 Serono established a master cell bank and working cell bank, from which all
cells originate, which produce recombinant human FSH (Gonal F®)
1990 First batch of recombinant human FSH made for clinical studies
1992 First pregnancy using recombinant human FSH following an IVF cycle in
Switzerland and Belgium
1995 Gonal F® is the first pharmaceutical to be granted European-wide marketing
approval by the European Medicines Evaluation Agency
1996 Puregon® is registered in Europe
1997 First pregnancy following use of only recombinant gonadotrophins
(recombinant human FSH, LH and HCG) in a woman suffering from WHO I
amenorrhoea (Agrawal et al., 1997)
2000 European registration of recombinant human LH (Luveris®)
2001 European registration of recombinant human HCG (Ovidrel®/Ovitrelle®)

Luveris® = recombinant human LH; Ovitrelle® = recombinant HCG.

recombinant human gonadotrophins, and both recombinant
human LH and recombinant human HCG are now available on
the market. Milestones in the development of the recombinant
human gonadotrophins are shown in Table 4.
Use of GnRH analogues in ovarian
stimulation procedures
For many years, premature LH surges leading to poor-quality
oocytes and embryos, and low implantation and pregnancy
rates, were a common complication of ovarian stimulation. In
1983, however, a GnRH agonist, buserelin, was used for the
first time to suppress endogenous LH surges according to a
treatment protocol termed the ’long GnRH agonist protocol’
(Porter et al., 1984). This protocol involves the administration
of a GnRH agonist starting on either the early follicular or the
mid-luteal phase of the preceding menstrual cycle. After 14
days, when pituitary LH secretion has been suppressed, the
patient receives gonadotrophins until the follicles reach an
appropriate size and HCG can be given to induce ovulation.
This protocol has now been used as the stimulation protocol of
choice for nearly two decades. In Germany, for example, it is
used in 65% of all IVF cycles.
GnRH antagonists have also been developed in parallel with
the GnRH agonists. At first, the former had the disadvantage
of stimulating histamine release, with the consequent problems
of allergic reactions. In, 1999, however, cetrorelix
(Cetrotide®, Serono International S.A., Geneva, Switzerland)
was introduced to the market originally by ASTA Medica
(Frankfurt, Germany), and this was followed a year later by
ganirelix (Orgalutran®, Antagon®, Organon, Oss, The
Netherlands). Neither of these GnRH antagonists significantly
stimulates histamine release. They are both administered once 81

Reprinted from Vol. 5, Suppl. 1 (2002) pp. 73 86. S11

 Ovarian stimulation: from basic science to clinical application - M Ludwig et al.
b surge for about 96 h.
daily at a dose of 0.25 mg (Figure 10). Cetrorelix is also
available as a 3 mg dose, allowing a single administration
during the mid-follicular phase which guarantees pituitary
suppression for 96 h (Chillik and Acosta, 2001; Howles,
2002).
The major advantages of using GnRH antagonists rather than
GnRH agonists for ovarian stimulation are discussed
extensively in other publications in this supplement (Ludwig et
al., 2002). Briefly, these include a shorter duration of
treatment, no flare-up effect and hence no risk of cyst
formation, no depletion of sex steroids and the consequent side
effects, a shorter period of ovarian stimulation and hence the
administration of a lower cumulative dose of gonadotrophins,
and a lower risk of OHSS. However, several studies have also
investigated the possibility of a lower pregnancy rate with
GnRH antagonists (Olivennes et al., 2000; Albano et al., 2000;
The European Orgalutran Study Group et al., 2000).
Further prospective, randomized studies are needed to
determine whether there are actually any differences between
the different GnRH antagonists or between GnRH agonists or
antagonists (Ludwig et al., 2001).
Recent advances in ovarian
stimulation
With the advent of recombinant human gonadotrophins,
ovarian stimulation procedures have become safer and more
convenient for the patient than ever before. In particular,
gonadotrophins can now be administered by subcutaneous
injection, which makes self-administration a viable option.
Patients can therefore administer their own treatment at home,
82 making it simpler, cheaper and more convenient.
S12
Figure 10. Ovarian stimulation using GnRH
antagonists. Shown are the multiple-dose (a) and the
single-dose (b) protocols. In the standardized
multiple dose protocol, gonadotrophin administration
starts on day 2 or 3 following spontaneous menstrual
bleeding. The GnRH antagonist is given starting on
day 6 of ovarian stimulation (0.25 cetrorelix or
ganirelix). It is administered up to and including the
day of HCG. In the single dose protocol (b) the
GnRH antagonist is given only once (3 mg
cetrorelix), on day 7 of ovarian stimulation, i.e. day 8
of the cycle. This will protect against a premature LH
Novel delivery systems
Follitropin alpha is available as a multidose presentation, one
ampoule containing the equivalent of fourteen 75 IU doses of
recombinant human FSH. Once reconstituted, this
gonadotrophin solution can be stored at room temperature for
up to 28 days (Munafo et al., 2001).
In the last 2 years, sophisticated injection methods have been
developed. The autoinjecting Puregon Pen® (follitropin beta
administered by a pen device, Organon, Oss, The
Netherlands), for example, allows the dose administered to be
altered in 25 IU increments, enabling the adjustment of the
required dose. The pen device for administering follitropin
beta developed from a device used for long-term delivery of
insulin to diabetic patients, and is a welcome addition to the
tools available to physicians treating infertility (Voortman et
al., 1999). Both follitropin beta administered by a pen device
and the multidose presentation of follitropin alpha allow the
injection of a very low volume of gonadotrophin, which
increases patient comfort during treatment.
New recombinant human FSH
formulation: follitropin alpha filled by mass
A new presentation of recombinant human FSH has recently
been developed by Serono S.A. This presentation, known as
Gonal F® filled by mass (FbM) (follitropin alpha FbM;
Serono International S.A., Geneva, Switzerland), differs from
conventional follitropin alpha in that the product is filled by
protein content (mass in micrograms) rather than by biological
activity. It is claimed that follitropin alpha FbM is produced
using an improved manufacturing process, which employs
physico-chemical techniques rather than an in-vivo bioassay to
Reprinted from Vol. 5, Suppl. 1 (2002) pp. 73 86.
 Ovarian stimulation: from basic science to clinical application - M Ludwig et al.
measure the amount (mass) of FSH protein added to each vial
during filling (Driebergen et al., 2002).
The biological activity of FSH derives from the sequence of
amino acids in its α- and β-chains, and from the specific
pattern of glycosylation. Different isoforms of FSH exist due
to different patterns of glycosylation of the parent molecule.
Sialic acid residues play a major role in protecting the
molecule against rapid metabolic clearance in vivo. For this
reason FSH isoforms show differences in in-vivo bioactivity
which are related to differences in isolelectric point (pI).
Isoforms with a pI around 3.5 are 100–200 times more potent
in the in-vivo bioassay than isoforms with a pI of 5.5–6.0.
Current preparations of recombinant human FSH contain a
mix of the different isoforms.
It is well recognized in the biopharmaceutical industry that the
use of an in-vivo bioassay to determine the biopotency of a
drug has many disadvantages: it requires the use of many
animals, typically 120–180 to obtain a single test result, and it
is imprecise, inaccurate and not environmentally friendly. The
coefficient of variation for an in-vivo bioassay is typically
around 10%, which cannot compare to the performance of
physico–chemical analytical techniques such as size exclusion
high-performance liquid chromatography (SE-HPLC), for
which the coefficient of variation is typically 2–3%. The new
manufacturing process for follitropin alpha FbM involves the
use of two physico-chemical techniques to ensure the
consistency of the preparations – glycan mapping and
isoelectric focusing. A highly precise SE-HPLC assay is used
to quantify accurately the drug substance content in the
product.
The glycan mapping method provides a fingerprint of the
glycan species of r-hFSH and an estimation of the degree of
sialylation of the oligosaccharide chains. After release from
the protein by hydrazinolysis, the intact glycan species are
labelled with a fluorescent derivative. Each glycan molecule is
labelled with a single molecule of dye reagent. The response
coefficient is therefore the same for all the glycan species. The
glycan species are then separated according to charge by anion
exchange chromatography, and detected by fluorimetry. Under
these conditions, the glycan species are eluted as a function of
their charge, which is related to the number of sialic acids they
carry. The results are expressed as the relative percentage of
the glycan species grouped as a function of their charge. A
hypothetical charge number Z has been defined as the sum of
the percent areas under the curve in the neutral, mono-, di-, tri-
and tetra-sialylated glycan regions multiplied by their
corresponding charge (Hermentin et al., 1996).
Isoelectric focusing (IEF) of r-hFSH is performed in a suitable
gel matrix across a pH range of 3.5–7 and visualized by
Coomassie Brilliant Blue staining. The gel is then scanned and
the pI values as well as band intensities of the sample isoforms
are compared with those obtained for the Reference House
Standard. It can therefore be concluded that the drug substance
produced by the commercial manufacturing process is highly
consistent in isoform distribution.
Use of these techniques enables the consistency of the
manufactured product to be guaranteed in terms of both
glycosylation pattern and isoform profile. Because of the
Reprinted from Vol. 5, Suppl. 1 (2002) pp. 73 86.
known relationship between isoform profile and activity, this
means that the calculated weight of FSH protein consistently
delivers the same biological activity. In recognition of this fact,
vials of follitropin alpha FbM are labelled by mass (and by
biological activity), and a SE-HPLC assay is used to test the
strength/potency of this product (Driebergen et al., 2002).
Clinical experience with follitropin alpha
FbM
In a multicentre, prospective, randomized, comparative trial,
456 women undergoing IVF were treated with either
follitropin alpha FbM (220 patients) or follitropin alpha (223
patients) according to a long luteal protocol for an average of
9.7 ± 1.8 and 10.2 ± 1.8 days respectively. The average number
of ampoules of follitropin alpha used was significantly lower
(26.1 ± 10.3) for patients treated with the FbM formulation
than for those treated with the standard formulation (29.3 ±
11.9; P < 0.01). The oestradiol concentration on the day of
HCG administration was significantly higher in the follitropin
alpha FbM group than in the group receiving conventional
follitropin alpha (7792 ± 4911 versus 6642 ± 4219 pmol/l; P <
0.01), and the number of retrieved oocytes (11.9 ± 6.3 versus
10.8 ± 6.8; P = 0.052) was greater. Consequently, the total
number of embryos obtained was also higher in the follitropin
alpha FbM group (6.5 ± 4.0 versus 5.7 ± 3.7; P < 0.05). The
researchers concluded that improvements in the manufacturing
process for recombinant human human FSH were responsible
for this increase in quality of the product (Abuzeid et al., 2001;
Neuspillier et al., 2002). This was confirmed by another
prospective, randomized, double-blind study. In this study
follitropin alpha, coming from four bulk lots of r-hFSH, was
filled to eight batches, one batch filled by the conventional
bioassay and one filled by mass, so that two batches came from
each bulk lot. The results showed more consistency and less
between-batch variation using the follitropin alpha FbM as
compared with the conventional follitropin alpha (Hugues et
al., 2002).
Long-acting recombinant human FSH
Two different ways of developing long-acting gonadotrophin
preparations are currently under investigation. Such
preparations should enable gonadotrophins to be injected only
once or twice during a stimulation cycle rather than daily, as is
currently the case.
The results achieved with a long-acting FSH molecule in an
international, multicentre trial were published recently
(Bouloux et al., 2001). This molecule consisted of the original
FSH α subunit and a hybrid β-subunit and was synthesized by
Organon (Oss, The Netherlands). The latter was made from the
original FSH molecule and the c-terminal peptide (CTP) from
HCG. The idea for this design came from the observation that
CTP is the main distinguishing feature between LH and HCG,
and may therefore be responsible for the prolonged half-life of
HCG. Indeed, this newly designed FSH molecule is reported to
have a half-life in animals and in-vivo bioactivity that are two
times greater than those of natural FSH (Bouloux et al., 2001).
When the molecule was administered to 13
hypogonadotrophic males, no drug-related adverse events
were observed, although symptoms of mild local tolerance
symptoms occurred in 39% of the subjects. These symptoms 83
S13
 Ovarian stimulation: from basic science to clinical application - M Ludwig et al.
lasted for more than 24 h in 31% of subjects. No antibodies to
either component of the FSH molecule were observed, and the
elimination half-life was reported to be 94.7 ± 26.2 h, which is
2–3 times longer than that of natural FSH (Bouloux et al.,
2001). The data from female patients have not yet been
published.
Serono International S.A. is also developing a novel, long-
acting formulation of recombinant human FSH (Dr Steve
Arkinstall, Serono International S.A., personal
communication). The new formulation is based on Alkermes’
ProLease injectable sustained-release drug delivery
technology, which involves the encapsulation of a drug into
small polymeric microspheres. These degrade slowly and
release the encapsulated drug at a controlled rate following
subcutaneous or intramuscular injection. The new formulation
is designed to offer patients the alternative of a single injection
rather than multiple daily injections. Data on this formulation
have yet to be published.
Non-peptide molecules
As knowledge about the activating sites of gonadotrophins and
GnRH analogues has increased, it has become possible to
create small, non-peptide molecules that induce signal
transduction without binding to the extracellular domains of
membrane proteins. Such molecules will ultimately be
converted into highly potent orally active therapeutic
preparations, and will replace the dimeric glycoprotein
hormones or will act as antagonists. A number of these
molecules are already being actively tested. It is therefore
feasible that the future of ovarian stimulation will be a simple
treatment schedule composed of different tablets, which have
to be taken at certain time points during the ovarian
stimulation cycle.
The future of ovarian stimulation
Over the next 5–10 years, it is envisaged that more
sophisticated use of GnRH antagonists will lead to their
widespread use in ovarian stimulation protocols. The
gonadotrophin of choice will be recombinant human FSH,
since it guarantees a consistent structure and biological
activity. Where necessary, LH can be added to the protocol by
the administration of recombinant LH, and ovulation will be
induced using recombinant HCG. Also, fixed combinations of
recombinant LH and recombinant human FSH may be an
attractive option.
Technology has come a long way since the era of animal-,
human pituitary- and urinary-derived hormones, and it seems
clear that new pharmaceutical products will offer the safest
option for ovarian stimulation during assisted reproductive
techniques.
References
Abuzeid MI, Kelly E, Loumaye E et al. 2001 A new formulation of
Gonal-F (r-hFSH) filled by mass delivers more and better oocytes
and embryos with a lower cumulative dose when compared with
the current follitropin alfa preparation in ovarian stimulation for
ART. Preliminary data. Middle East Fertility Society Journal 6,
O33.
84
S14
Agrawal R, West C, Conway GS et al. 1997 Pregnancy after
treatment with three recombinant gonadotropins. Lancet 349,
29–30.
Albano C, Felberbaum RE, Smitz J et al. 2000 Controlled ovarian
stimulation with HMG: results of a prospective randomized phase
III European study comparing the LHRH-antagonist Cetrorelix
(Cetrotide) and the LHRH-agonist Buserelin. Human
Reproduction 15, 526–531.
Aschner B 1912 Ueber die Beziehung zwischen Hypophysis und
Genitale. Archives of Gynecology 97, 200–227.
Beclere A 1926 [Article title unavailable]. Paris Medical 13, 97.
Bettendorf G 1963 Human hypophyseal gonadotropin in
hypophysectomized women. International Journal of Fertility 8,
799.
Borth R, Lunenfeld B, de Watteville H 1954 Activité gonadotrope
d’un extrait d’urines de femmes en menopause. Experientia 10,
266–270.
Bouloux PM, Handelsman DJ, Jockenhovel F et al. 2001 First
human exposure to FSH-CTP in hypogonadotrophic hypogonadal
males. Human Reproduction 16, 1592–1597.
Chillik C, Acosta A 2001 The role of LHRH agonists and
antagonists. Reproductive BioMedicine Online 2, 120–128.
Cluroe AD, Synek BJ 1995 A fatal case of ovarian hyperstimulation
syndrome with cerebral infarction. Pathology 27, 344–346.
Cochius JI, Burns RJ, Blumbergs PC et al. 1990 Creutzfeldt–Jakob
disease in a recipient of human pituitary-derived gonadotrophin.
Australia and New Zealand Journal of Medicine 20, 592–593.
Cole HH, Hart GH 1930 The potency of blood serum of mares in
progressive stages of pregnancy in effecting the sexual maturity
of the immature rat. American Journal of Physiology 93, 57–68.
Crowe SJ, Cushing H, Homans J 1910 Experimental
hypophysectomy. Bulletin of the Johns Hopkins Hospital 21,
127–167.
Cummins J 1999 Evolutionary forces behind human infertility.
Nature 397, 557–558.
Daya S 2002 Updated meta-analysis of recombinant follicle-
stimulating hormone (FSH) versus urinary FSH for ovarian
stimulation in assisted reproduction. Fertility and Sterility 77,
711–714.
Daya S, Gunby J, Hughes EG et al. 1995 Follicle-stimulating
hormone versus human menopausal gonadotropin for in vitro
fertilization cycles: a meta-analysis. Fertility and Sterility 64,
347–354.
Daya S, Ledger W, Auray JP et al. 2001 Cost-effectiveness
modelling of recombinant FSH versus urinary FSH in assisted
reproduction techniques in the UK. Human Reproduction 16,
2563–2569.
Donini P, Puzzuoli D, Montezemolo R 1964 Purification of
gonadotropin from human menopausal urine. Acta
Endocrinologica 45, 329.
Donini P, Puzzuoli D, D’Alessio C et al. 1966 Purification and
separation of follicle-stimulating hormone (FSH) and luteinizing
hormone (LH) from human postmenopausal gonadotrophin
(HMG). Acta Endocrinologica 52, 169–185.
Driebergen R, Bassett R, Baer G et al. 2002 Improvements in
quantification of recombinant human FSH activity: SE-HPLC
versus the in-vivo rat bioassay. Human Reproduction (Abstract
book) 17, 163.
Dumble LJ, Klein RD 1992 Creutzfeldt–Jakob legacy for Australian
women treated with human pituitary gonadotropins. Lancet 340,
847–848.
Eshkol A, Lunenfeld B 1967 Purification and separation of follicle
stimulating hormone (FSH) and luteinizing hormone (LH) from
human menopausal gonadotrophin (HMG). Acta Endocrinologica
54, 919–927.
Fauser BC, Donderwinkel P, Schoot DC 1993 The step-down
principle in gonadotrophin treatment and the role of GnRH
analogues. Baillieres Clinical Obstetrics and Gynaecology 7,
309–330.
Fevold HL, Hisaw FL, Leonard SL 1931 The gonad stimulating and
Reprinted from Vol. 5, Suppl. 1 (2002) pp. 73 86.
 Ovarian stimulation: from basic science to clinical application - M Ludwig et al.
the luteinizing hormones of the anterior lobe of the pituitary.
American Journal of Physiology 109, 655–665.
Finkler RS 1949 Evaluation of hormonal and radiation therapy in,
190 cases of functional sterility and secondary amenorrhea.
Preliminary report. American Journal of Obstetrics and
Gynecology 58, 559–564.
Gemzell CA, Diszfalusy E, Tillinger, G 1958 Clinical effect of
human pituitary follicle stimulating hormone. Journal of Clinical
Endocrinology and Metabolism 18, 138–148.
Giudice E, Crisci C, Altarocca V, O’Brien M 2001 Characterization
of a partially purified human menopausal gonadotropin
preparation. Journal of Clinical Research 4, 27–34.
Groot-Wassink K, Blawert H 1973 Vergleichende Untersuchungen
zur Ovulationsauslösung mit Follistiman und Pergonal.
Zentralblatt for Gynakologie 9, 1019–1024.
Guillemin R 1967 Chemistry and physiology of hypothalamic
releasing factors for gonadotrophins. International Journal of
Fertility 12, 359–367.
Halberstaedter L 1905 [Article title unavailable]. Berlin Klinische
Wochenschrift 42, 64.
Hermentin P, Witzel R, Kanzy E-J et al. 1996 The hpothetical N-
glycan charge: a number that characterizes protein glycosylation.
Glycobiology 6, 217–230.
Howles CM 1996 Genetic engineering of human FSH (Gonal-F).
Human Reproduction Update 2, 172–191.
Howles CM 2002 The place of gonadotrophin-releasing hormone
antagonists in reproductive medicine. Reproductive BioMedicine
Online 4, 64–71.
Howles C, Loumaye E, Giroud D, Luyet G 1994 Multiple follicular
development and ovarian steroidogenesis following subcutaneous
administration of a highly purified urinary FSH preparation in
pituitary desensitized women undergoing IVF: a multicentre
European phase III study. Human Reproduction 9, 424–430.
Hugues JN 2001 Recombinant human follicle-stimulating hormone: a
scientific step to clinical improvement. Reproductive BioMedicine
Online 2, 54–64.
Hugues JN, Cedrin-Durnerin I, Avril C et al. 1996 Sequential step-up
and step-down dose regimen: an alternative method for ovulation
induction with follicle-stimulating hormone in polycystic ovarian
syndrome. Human Reproduction 11, 2581–2584.
Hugues JN, Barlow DH, Rosenwaks Z et al. 2002 A new filled-by-
mass manufacturing process for Gonal F delivers a more
consistent clinical response. Human Reproduction (Abstract
book) 17, 88.
Kaplan I 1948 The use of high voltage roentgentherapy in the
treatment of amenorrhea and sterility in women. American
Journal of Roentgenology 59, 3703–3779.
Knobil E 1974 On the control of gonadotropin secretion in the rhesus
monkey. Recent Progress in Hormone Research 30, 1–46.
Knobil E 1980 The neuroendocrine control of the menstrual cycle.
Recent Progress in Hormone Research 36, 53–88.
Knobil E 1988 The neuroendocrine control of ovulation. Human
Reproduction 3, 469–472.
le Cotonnec JY, Porchet HC, Beltrami V, Howles C 1993
Comparative pharmacokinetics of two urinary human follicle
stimulating hormone preparations in healthy female and male
volunteers. Human Reproduction 8, 1604–1611.
Ludwig M, Felberbaum RE, Diedrich K 2000 Deep vein thrombosis
during administration of HMG for ovarian stimulation. Archives
of Gynecology and Obstetrics 263, 139–141.
Ludwig M, Finas DF, Al-Hasani S et al. 1999a Oocyte quality and
treatment outcome in intracytoplasmic sperm injection cycles of
polycystic ovarian syndrome patients. Human Reproduction 14,
354–358.
Ludwig M, Katalinic A, Diedrich K 2001 Use of GnRH antagonists
in ovarian stimulation for ART compared to the long protocol: a
meta-analysis. Archives of Gynecology and Obstetrics 265,
175–182.
Ludwig M, Katalinic A, Felberbaum RE, Diedrich K 2002 Safety
aspects of GnRH antagonists in ovarian stimulation procedures:
Reprinted from Vol. 5, Suppl. 1 (2002) pp. 73 86.
ovarian hyperstimulation syndrome and health of children born.
Reproductive BioMedicine Online 5 (suppl. 1), 61–67.
Ludwig M, Tölg R, Richardt G et al. 1999b Myocardial infarction
associated with ovarian hyperstimulation syndrome. JAMA 282,
632–633.
Lunenfeld B 1963 Treatment of anovulation by human
gonadotropins. International Journal of Obstetrics and
Gynecology 1, 153.
Lunenfeld B 1995 Infertility throughout the ages. In: AT Alberda, RA
Gan, HM Vemer (eds) Proceedings of a symposium held in Oss,
The Netherlands, 1993. Parthenon Publishing Group, New York,
London, pp. 55–71.
Lunenfeld B, Menzi A, Volet B 1960 Clinical effects of human post-
menopausal gonadotrophin. In: F Fuchs (ed.) Advance Abstracts
of Short Communications. First International Congress of
Endocrinology, Copenhagen, p. 587.
Lunenfeld B, Rabau E, Rumney G, Winkelsberg G 1961 The
responsiveness of the human ovary to gonadotrophin. (Hypophsis
III). Proceedings of Third World Congress Gynecology and
Obstetrics, Vienna 1, 220.
Lunenfeld B, Sulimovici S, Rabau E, Eshkol A 1962 L’induction de
l’ovulation dans les amenorrheas hypophysaires par un traitement
combine de gonadotrophins urinaires menopausiques et de
gonadotrophins chorioniques. Comptes Rendus de la Société
Française de Gynecologie 32, 346.
Mazer C 1946 Diagnosis and Treatment of Menstrual Disorders and
Sterility. Paul B. Hoeber, New York.
Mazer C, Greenberg R 1943 Low dosage irradiation in the treatment
of amenorrhea. American Journal of Obstetrics and Gynecology
46, 648–652.
Melmed H, Mashiach S, Insler V et al. 1969 The response of the
hyposensitive ovary to massive stimulation with human
gonadotrophins. Journal of Obstetrics and Gynaecology of the
British Commonwealth 76, 437–443.
Mozes M, Bogokowsky H, Antebi E et al. 1965 Thromboembolic
phenomena after ovarian stimulation with human gonadotrophins.
Lancet 2, 1213–1215.
Munafo A, Bilham W, Trinchard-Lugan I, Buraglio M 2001
Comparative bioavailability and safety of a single dose and a
multiple dose formulation of recombinant human follicle
stimulating hormone (Gonal-F). Journal of Clinical Research 4,
12–26.
Neuspillier N, Kelly E, Denton G et al. 2002 Technical
improvements in the Gonal F (filled by mass) manufacturing
process yield improved clinical results. Human Reproduction
(Abstract book) 17, 71–72.
Olivennes F, Belaisch-Allart J, Emperaire JC et al. 2000 A
prospective randomized controlled study in IVF-ET with a single
dose of a LH-RH antagonist (Cetrorelix) or a depot formula of a
LH-RH agonist (Triptorelin). Fertility and Sterility 73, 314–320.
Ostergaard E 1942 Antigonadotrophic Substances. Munksgaard,
Copenhagen, Denmark.
Plant TM, Nakai Y, Belchetz P et al. 1978 The sites of action of
estradiol and phentolamine in the inhibition of the pulsatile,
circhoral discharges of LH in the rhesus monkey (Macaca
mulatta). Endocrinology 102, 1015–1018.
Porter RN, Smith W, Craft IL et al. 1984 Induction of ovulation for
in-vitro fertilisation using buserelin and gonadotropins. Lancet 2,
1284–1285.
Risquez F 2001 Induction of follicular growth and ovulation with
urinary and recombinant gonadotrophins. Reproductive
BioMedicine Online 3, 54–72.
Rodgers M, McLoughlin J, Peers N et al. 1994 Accumulation of
human chorionic gonadotrophin in the serum of patients during
in-vitro fertilization treatment cycles with Pergonal. Human
Reproduction 9, 638–642.
Ron E, Boice JD Jr, Hamburger S, Stovall M 1994 Mortality
following radiation treatment for infertility of hormonal origin or
amenorrhoea. International Journal of Epidemiology 23,
1165–1173.
85
S15
 Ovarian stimulation: from basic science to clinical application - M Ludwig et al.
Rubin IC 1926 Sterility associated with habitual amenorrhea relieved
by X ray therapy. American Journal of Obstetrics and
Gynecology 12, 76–82.
Ryan KJ 1979 Granulosa–theca cell interaction in ovaria. Journal of
Steroid Biochemistry 11, 799–800.
Ryan KJ, Petro Z 1966 Steroid biosynthesis by human ovarian
granulosa and theca cells. Journal of Clinical Endocrinology
Metabolism 26, 46–51.
Schally AV, Nair RM, Redding TW, Arimura A 1971 Isolation of the
luteinizing hormone and follicle-stimulating hormone-releasing
hormone from porcine hypothalami. Journal of Biological
Chemistry 246, 7230–7236.
Silverberg K, Daya S, Auray JP et al. 2002a Analysis of the cost
effectiveness of recombinant versus urinary follicle-stimulating
hormone in in vitro fertilization/intracytoplasmic sperm injection
programs in the United States. Fertility and Sterility 77, 107–113.
Silverberg K, Schertz J, Falk B, Beresniak A 2002b Impact of urinary
FSH price: a cost-effectiveness analysis of recombinant and
urinary FSH in assisted reproduction techniques in the USA.
Reproductive BioMedicine Online 5, 265–269.
Smith PE 1926 Hastening of development of female genital system
by daily hemoplastic pituitary transplants. Proceedings of the
Society for Experimental Biology and Medicine 24, 1311–1333.
Smith PE, Engle ET 1927 Experimental evidence of the role of
anterior pituitary in development and regulation of gonads.
American Journal of Anatomy 40, 159.
Snick HK, Snick TS, Evers JL, Collins JA 1997 The spontaneous
pregnancy prognosis in untreated subfertile couples: the
Walcheren primary care study. Human Reproduction 12,
1582–1588.
Stanger JD, Yovich JL 1985 Reduced in vitro fertlization of human
oocytes from patients with raised basal luteinizing hormone
levels during the follicular phase. British Journal of Obstetrics
and Gynaecology 92, 385–393.
Steptoe PC, Edwards RG 1976 Reimplantation of a human embryo
with subsequent tubal pregnancy. Lancet 1, 880–882.
Steptoe PC, Edwards RG 1978 Birth after the reimplantation of a
human embryo. Lancet 2, 366.
Stokman PG, De Leeuw R, van den Wijngaard HA et al. 1993
Human chorionic gonadotropin in commercial human
menopausal gonadotropin preparations. Fertility and Sterility 60,
175–178.
Sykes D, Out HJ, Palmer SJ, van Loon J 2001 The cost-effectiveness
of IVF in the UK: a comparison of three gonadotrophin
treatments. Human Reproduction 16, 2557–2562.
The European Orgalutran Study Group, Mannaerts BMJL, Borm G
86
S16
2000 Treatment with the gonadotrophin-releasing hormone
antagonist ganirelix in women undergoing ovarian stimulation
with recombinant follicle stimulating hormone is effective, safe
and convenient: results of a controlled, randomized, multicentre
trial. Human Reproduction 15, 1490–1498.
The European Recombinant Human LH Study Group 1998
Recombinant human luteinizing hormone (LH) to support
recombinant human follicle-stimulating hormone (FSH)-induced
follicular development in LH- and FSH-deficient anovulatory
women: a dose-finding study. The European Recombinant Human
LH Study Group. Journal of Clinical Endocrinology and
Metabolism 83, 1507–1514.
Voortman G, van de Post J, Schoemaker RC, van Geerven JMA 1999
Bioequivalence of subcutaneous injections of recombinant human
follicle stimulating hormone (Puregon(R)) by Pen-injector and
syringe. Human Reproduction 14, 1698–1702.
Wetsel WC, Liposits Z, Seidah NG, Collins S 1995 Expression of
candidate pro-GnRH processing enzymes in rat hypothalamus
and an immortalized hypothalamic neuronal cell line.
Neuroendocrinology 62, 166–177.
Wetsel WC, Mellon PL, Weiner RI, Negro-Vilar A 1991 Metabolism
of pro-luteinizing hormone-releasing hormone in immortalized
hypothalamic neurons. Endocrinology 129, 1584–1595.
World Health Organization 1973 Agents stimulatins gonadal function
in human. World Health Organization Technical Report Series,
pp. 514–520.
Wildt L, Hausler A, Marshall G et al. 1981 Frequency and amplitude
of gonadotropin-releasing hormone stimulation and gonadotropin
secretion in the rhesus monkey. Endocrinology 109, 376–385.
Wildt L, Diedrich K, van der Ven HH et al. 1986 Ovarian
hyperstimulation for in-vitro fertilization controlled by GnRH
agonist administered in combination with human menopausal
gonadotrophins. Human Reproduction 1, 15–19.
Yen SSC 1999 Neuroendocrinology of reproduction. In: SSC Yen,
RB Jaffe, RL Barbieri (eds) Reproductive Endocrinology.
Saunders, Philadelphia, London, Toronto, Montreal, Sydney,
Tokyo, pp. 30–80.
Zondek B 1926 Ueber die Funktion des Ovariums. Zeitschrift für
Geburtshilfe und Gynäkologie 90, 327.
Zondek B 1930 Ueber die Hormone des Hypophysenvorderlappens.
Klinische Wochenschrift 9.
Zondek B, Aschheim S 1927 Das Hormon des
Hypophysenvorderlappens: Testobjekt zum Nachweis des
Hormons. Klinische Wochenschrift 6, 248–254.
Zondek B, Sulman F 1942 The Antigonadotropic Factor. Williams
Wilkinsm Baltimore.
Reprinted from Vol. 5, Suppl. 1 (2002) pp. 73 86.