Clinical efficacy of recombinant gonadotrophins

Adam H. Balen1, Catherine J. Hayden and Anthony J. Rutherford

Department of Reproductive Medicine, Clarendon Wing, Leeds General Infirmary, Leeds LS2 9NS, UK

The advent of recombinantly-derived gonadotrophin preparations has been heralded as a major breakthrough in the therapeutic armory of assisted reproduction treatments (McDonough, 1995Go). Godwin Meniru poses a timely question by asking whether the new generation of drugs is providing any real benefit (Meniru, 1999Go). To date there are two recombinant follicle stimulating hormone (rFSH) preparations: follitropin {alpha} (Gonal-F; Serono Laboratories) and follitropin ß (Puregon, Organon Laboratories). In discussing the benefits of a gonadotrophin preparation, one has to consider clinical efficacy, side-effects and cost-effectiveness. Clinical efficacy includes the ability to stimulate folliculogenesis, the production of mature oocytes, appropriate steroidogenesis for endometrial development and, in the context of in-vitro fertilization (IVF), sufficient quality pre-embryos and, ultimately, good pregnancy rates. Before moving on to these fundamental issues, we would like first to discuss the particular points raised by the first paper in this debate (Meniru, 1999Go).

Meniru (1999) cites another paper (Jacob et al., 1998Go) and uses it to damp down the enthusiastic initial industry-sponsored reports on the efficacy of rFSH preparations. We would suggest, however, that Jacob's paper should not have been published. Jacob et al. (1998) retrospectively analysed the last 191 women who had received human menopausal gonadotrophin (HMG) in their IVF programme and compared them with the first 162 women to receive rFSH. Different urinary gonadotrophin preparations were used and some patients received growth hormone in addition; different starting doses were also used depending upon baseline serum FSH concentrations and response in previous cycles. Furthermore, 150 IU rFSH was considered to be equivalent to 225 IU HMG, based on the recommendations of Out et al. (1995), even though the study by Out did not set out to demonstrate equivalence. Suffice it to say therefore that a retrospective analysis of a heterogeneous data set would be considered by many to be meaningless and does not really warrant further consideration.

Meniru himself (1999) uses data from 13 patients given Puregon at different starting doses and reports differing responses and variable requirements to either increase or decrease the dose. He also states that six patients found the Puregon injections to be more painful than Profasi (human chorionic gonadotrophin; HCG), although this was not studied in any formalized way. Such anecdotal reporting is unscientific and, frankly, unhelpful. If we now move away from the examples cited by Meniru and discuss the broader issues there are indeed a number of points that are worthy of debate.

Historical perspective

The original sources of gonadotrophins for therapeutic use were post-mortem pituitary extracts and the urine of post-menopausal women. The former source was withdrawn because of cases of Creutzfeld–Jakob disease, which occurred predominantly in Australia, but also in Europe. The extraction and purification of post-menopausal urine was pioneered in Italy in the late 1940s to result in the production of HMG (Donini et al., 1949). A total of 20–30 l of post-menopausal urine was required to treat one patient with one cycle of HMG. Through the 1960s the extraction process to remove non-specific co-purified proteins became more sophisticated such that activity was increased 10-fold over the early preparations to 100–150 IU FSH/mg protein. Greater purity produced fewer hypersensitivity reactions and less discomfort from the smaller volume of the injection. Despite the vastly increased purity of HMG (menotropin) and uFSH (urofollitropin) compared with the original preparations, their active ingredients only made up 1–2 % of the final product. They still contain large amounts of urinary protein (including cytokines, growth factors, transferrins and other proteins that might modulate ovarian activity) which makes uniform standardization very difficult, leads to local reactions at the injection sites and occasionally systemic illness. The advent of monoclonal antibodies in the 1980s, enabled further purification to be achieved by specifically selecting FSH out from the bulk HMG (Howles et al., 1992Go). The extract was 95% pure with a several hundred fold enhancement of specific gonadotrophin bioactivity and known as `highly purified urinary FSH' (u-hFSH HP, Metrodin HP®; Serono Laboratories, Aubonne, Switzerland). Extended clinical trials comparing uFSH (urofollitropin) and highly purified FSH demonstrated equivalent ovulation and pregnancy rates. Much reduced hypersensitivity was reported such that the subcutaneous route could be adopted for administration (Dore et al., 1994Go; Howles et al., 1994Go; Wickland et al., 1994). However, the problems of supply, collection, transport, storage and processing of an ever-increasing requirement of urine remained and the pharmaceutical companies began to explore the emerging new technology of genetic engineering. The two genes for the {alpha} and ß subunits of FSH were incorporated into vectors for introduction into cells from the Chinese hamster ovary cell line and hereby began the process that should, theoretically result in an unlimited supply (Hayden et al., 1999).

It is the degree of glycosylation that affects the biological activity of FSH (Rose et al., 1999Go). This can only be measured by bioassay and is not measurable by immunoassay. Pharmacopoeial monographs taking account of the inherent precision of the methods in bioassays allow 95% confidence limits of 80–125% of the stated dose on estimates of activity, i.e. 60–94 units of activity in a 75 unit ampoule (a potential variation of up to 57% between ampoules from different batches). The same pharmacopeial requirements apply to the recombinantly-derived FSH preparations. There is evidence that there is heterogeneity between the different recombinantly-derived preparations, hence the nomenclature `follitropin {alpha} and ß'. The relationship of isoform composition to function has been recently reviewed (Rose et al., 1999Go). Data from in-vivo bioassays suggests that one of the major factors which controls FSH action is the relative degree of clearance of different isoforms. It is interesting to note that those forms of FSH which are most potent in vitro, tend to be least potent in vivo (Rose et al., 1999Go).

A large number of intrinsic and extrinsic factors affect the performance of a drug in vivo. In the case of rFSH, the pattern of glycosylation, specifically terminal sialylation of the protein backbone, has excited a great deal of interest, as it is crucial to the bioactivity of the hormone. Overall the isohormone composition of rFSH has proved to be very similar to pituitary extract but great effort has been spent establishing which forms have greatest bioactivity (Lambert et al., 1995Go) in order to design the most specific and predictable drug. Sialylation determines acidity and isoelectric charge. Basic forms have higher receptor binding activity and intrinsic bioactivity but are cleared more rapidly than acidic forms. The more acidic isoforms have a 20-fold higher in-vivo bioactivity, mainly due to their higher absorption, lower clearance rate and longer elimination half-life. The pharmacokinetics of rFSH (Gonal-F®) are very similar to uFSH (Metrodin®) (Le Cotonnec et al., 1994aGo,bGo). In the future it is not unreasonable to foresee modifications to the molecular structure that lead to an extension of the half-life and in-vivo bioactivity. This could enable the frequency of injections to be reduced, which would be greatly appreciated by the patient.

Advantages and disadvantages of rFSH

There are a number of immediately apparent advantages of rFSH over its urinary predecessors. Aside from the improved logistics of the pharmaceutical process, controlled manufacture has undoubtedly led to a more homogeneous product with much reduced inter-batch variability compared with the purification of enormous quantities of heterogeneous urine (Loumaye et al., 1995Go). The supply is potentially unlimited and shortages should no longer be a threat to clinical practice. There is no risk of infection or contamination with drugs or their metabolites as there is with products from a human source. The manufacturers have also confirmed that there have been no reported cases of seroconversion to anti-gonadotrophin antibodies (Recombinant Human FSH Study Group, 1995Go; Out et al., 1996Go).

The purity of the products has certainly enhanced their administration which is effective, safe and much less traumatic when the s.c. route is adopted (Out et al., 1995Go; Albano et al., 1996Go). The most obvious advantages of rFSH are greater purity and specificity. It has been inferred that smaller doses and a more predictable response will result (Bergh et al., 1997Go). The most dramatic disadvantage to the health provider and patient however, is a marked increase in the price of the product. And herein lies the biggest question: are the recombinant preparations more cost effective?

Assessment of clinical practice using recombinant human gonadotrophins for ovulation stimulation and ovulation induction

Research to date has been focused on comparing follitropin {alpha} (Gonal-F®; Serono) follitropin ß (Puregon®; Organon) with urinary preparations of FSH. They have not been compared with each other directly, although any differences that might exist between them are likely to be subtle.

IVF
Clinical evidence followed in therapeutic trials for IVF suggesting that rFSH (Puregon®) yields more oocytes, embryos and on-going pregnancies with a smaller dose for a shorter time than uFSH (Metrodin®) (Out et al., 1995Go). Frozen–thawed embryos were included in the figures as they resulted from the stimulation cycle involving the gonadotrophin in question. On-going pregnancy rates (at 12 weeks gestation) were consistently higher in the rFSH treated patients but not significantly so. Embryo quality appeared to be improved and having a surplus of embryos from which to choose the best to transfer increased the number available to be frozen. It is interesting to note that an initial comparative study of rFSH (Gonal-F®) with uFSH failed to show a significant difference in the number of retrieved or fertilized oocytes (Recombinant Human FSH study Group, 1995Go). The duration of treatment and the average dose required were also similar as were the numbers of patients reaching embryo transfer and implantation (clinical pregnancy). However, a later and larger comparative study of rFSH (Gonal-F®) with uFSH HP (Metrodin HP®) did reveal significant differences in the number of oocytes recovered (increased) and the number of FSH treatment days (reduced) (Bergh et al., 1997Go). The study included patients undergoing intracytoplasmic sperm injection (ICSI) where oocyte maturation is assessed prior to injection. There was no significant difference in oocyte maturity in the two groups. The clinical pregnancy rate for those patients who reached embryo transfer was similar for both groups suggesting no difference in embryo quality. This is interesting because pregnancy rates tend to be greater if there is a larger embryo pool from which to select for transfer (Staessen et al., 1993Go).

Because of the suggestion of increased potency of rFSH, NV Organon have chosen to market Puregon® in 50 and 100 IU ampoules (rather than the 75 IU and 150 IU previously chosen). Using multiple or fractions of ampoules is commonplace and contributes to the potential inaccuracy of the actual dose administered (especially in the inexperienced hands of the self-injecting patient). No trials have yet shown conclusively that 50 IU Puregon® is equivalent to 75 IU rFSH (Gonal F®) or uFSH. Furthermore it is essential to avoid confusion in prescribing practices and refer to doses in terms of IU and not in terms of multiples of ampoules.

Patients with polycystic ovaries certainly benefit from a reduced starting dose because of their propensity to an exuberant response to stimulation and increased risk of ovarian hyperstimulation syndrome (OHSS) (MacDougall et al., 1993Go). In the studies of rFSH to date, no significant differences in the incidence OHSS were found; however, since rFSH appears to be more potent, patients may be at greater risk thus vigilant monitoring remains essential in preventing this potentially fatal condition.

Fractions of ampoules have been employed for both ovulation stimulation regimens and also ovulation induction for anovulatory infertility. It can be difficult to accurately dilute the contents of an ampoule to provide a precise dose when using fractions (e.g. 1.5 75 IU ampoules should provide 112.5 IU) and the ability to start with doses of either 50 IU or 100 IU may be advantageous for patients with polycystic ovaries (PCO) (Devroey et al., 1998Go; Hayden et al., 1999aGo). Ultimately the pharmaceutical companies are planning to move away from lyophilized preparations to ampoules containing solutions that can be better titrated to the individual's needs by giving more precise incremental doses (H.J.Out, personal communication). Such formulations might certainly prove to be advantageous to the patient prone to overstimulation. The advantage here of the recombinant preparations is their stability in solution and this is a significant advance over the urinary preparations which, whilst maintaining immunoactivity over time, have been shown to have declining bioactivity in storage (Braileanu et al., 1998Go).

Very few studies have been performed comparing rFSH with menotropin, HMG which contains urinary contaminants and high concentrations of luteinizing hormone (LH). Favourable comparisons in terms of numbers of oocytes retrieved and on-going pregnancy rates with rFSH were demonstrated (Jansen and Van Os, 1996Go) but the study numbers were too small to reach statistical significance (89 women treated). However, a meta-analysis of urinary FSH with HMG has demonstrated that a significantly higher clinical pregnancy rate appears to be achieved with uFSH (Daya et al., 1995Go). This study implied that an adequate concentration of endogenous LH exists to achieve follicular and endometrial maturation, despite down-regulation of the pituitary with a gonadotrophin-releasing hormone (GnRH) analogue. Moreover, it has been suggested that exogenous LH supplementation in the form of HMG may be detrimental to the chances of achieving a pregnancy (Loumaye et al., 1996Go).

If, for the reasons described above, it appears that rFSH is inherently better than urinary-derived products, why should it be necessary to compare the two? The issue here is cost-effectiveness. In the UK and some other European countries, the urinary preparations are being phased out to make way for recombinant FSH. The costs of the preparations have increased exponentially. There are a number of new players in the pharmaceutical arena, the most notable in the UK, being Ferring with their HMG preparation Menogon®. The costs of the drugs has led to many clinics using Menogon with little published data of its efficacy, although it is certainly our clinical experience that the drug performs satisfactorily (unpublished data). We therefore feel that there is a need for a prospective randomized, assessor-blind study of HMG versus rFSH. If one sets significant levels at 5% with a power calculation based on a probability level of 80%, then assuming that a difference of <5% (for example, between a clinical pregnancy rate of 20 ± 5%) is not clinically significant we have calculated that 900 patients would be required in each arm of the study to prove the null hypothesis, i.e. that no difference exists between two preparations. We are co-ordinating a multi-centre study of this size in order to answer this very important clinical question.

Ovulation induction
Discussion has so far been confined to clinical studies of patients undergoing ovarian hyperstimulation for IVF. Gonadotrophins are also widely used for the induction of ovulation of a very small number of follicles (preferably one) in patients with WHO group II anovulation (mainly PCOS). Treatment strategies vary considerably, but as for IVF, there have been a few suggestions that rFSH improves outcome. It has been clearly demonstrated that co-administration of LH is not required to achieve follicular growth (Hayden et al., 1999bGo) and moreover LH does not influence the FSH threshold required to initiate follicular growth (van Weissenbruch et al., 1993Go). The role of LH in the management of infertility is a controversial one. Tonic hypersecretion of LH only occurs in PCOS but has been associated with increased infertility and miscarriage rates in this group (Balen et al., 1993Go). Conclusive prospective trials are still awaited as to whether the suppression of LH improves the outcome in these cases. The LH content of HMG does not significantly increase serum LH to pathological concentrations (Balen, 1999Go). The `purified' FSH preparations, or those with a reduced LH content, however, confer no therapeutic advantage over HMG as the LH content in HMG is trivial compared with the endogenous secretion of LH (Jacobs et al., 1987Go; Homburg et al., 1990a; Sagle et al., 1991Go; Fulghesu et al., 1992Go). Furthermore, pharmacokinetic studies do not indicate a significant difference when HMG and FSH are administered (Venturoli et al., 1986Go). It is also our experience that serum LH concentrations usually fall in response to normal ovarian–pituitary feedback as the dominant follicle grows, although some women with PCOS continue to oversecrete LH in the presence of follicular growth – a phenomenon that may be due to disordered production of non-steroidal ovarian factors in these patients (Balen and Rose, 1994Go).

The 2-cell two-gonadotrophin theory that granulosa cells require thecal cells, FSH and LH in order to produce oestrogen in the ovary has been well substantiated. Whilst some patients with hypothalamic-pituitary dysfunction (WHO group I anovulation) will respond to FSH alone (Schoot et al., 1992Go), the majority will not (Shoham et al., 1991bGo). Furthermore, even when a follicle does grow in response to FSH it produces a lower concentration of oestrogen and consequently a thinner endometrium. Thus for these patients LH-containing preparations are required.

In order to prevent the risks of overstimulation and multiple pregnancy in patients with PCOS, the traditional standard step-up regimens (Lunenfeld and Insler, 1974Go; Wang and Gemzell, 1980Go) have been replaced by either low-dose step-up regimens (Brown et al., 1969Go; Hamilton-Fairley et al., 1991Go; Shoham et al., 1991aGo; Balen et al., 1994Go) or step-down regimens (Fauser et al., 1993Go). Experimental studies have indicated that initiation of follicular growth requires a 10–30% increment in the dose of exogenous FSH and the threshold changes with follicular growth, due to an increased number of FSH receptors, so that the concentration of FSH required to maintain growth is less than that required to initiate it. The process of ovarian stimulation needs to be more controlled than for IVF as it is vital not to produce too many follicles if high order multiple pregnancy is to be avoided. It has been suggested that the greater purity and specificity of rFSH may help to control and predict ovarian response more accurately, ultimately leading to a greater percentage of successful cycles in terms of ovulation achieved, and fewer abandoned due to over-response. It can be extremely difficult to predict the response to stimulation of a women with polycystic ovaries – indeed this is the greatest therapeutic challenge in all ovulation induction therapies. The polycystic ovary is characteristically quiescent – at least when viewed by ultrasound – before often exhibiting an exuberant and explosive response to stimulation.

To date, there has been little evidence to suggest that rFSH is significantly better than uFSH in terms of pregnancy rates but rFSH (Puregon®) appears to be more efficient then uFSH (Metrodin®), in terms of dosage requirement and duration of stimulation in ovulation induction programs (Coelingh Bennink et al., 1998Go). We have recently published the first observational study of the use of rFSH (Puregon®) in the induction of ovulation in 11 patients with clomiphene resistant polycystic ovary syndrome (PCOS) (Hayden et al., 1999) and demonstrated that rFSH can be used successfully to stimulate follicular growth at a starting dose of 50 IU. Further refinement of protocols is still required and the advent of rFSH in solution might further enable close titration of dose per individual.

Future prospects

The field of genetic engineering will have much more to offer in the years to come. Recombinantly derived LH should enable a more physiological ovulatory trigger to be administered than the long-acting HCG currently employed. Recombinant LH will also find applications in ovulation induction/oestrogen generation for the patient with hypogonadotrophic hypogonadism (Kousta et al., 1996Go; European Recombinant LH Study Group, 1998).

In 1992, a new follitropin with a long half-life created by recombinant technology was reported (Fares et al., 1992Go). The longer half-life of HCG compared to LH was recognized as being a function of the extra 30 amino acids and carbohydrate residues located at the carboxyl terminal which did not impede interaction with the LH receptor but did lead to persistence in the circulation and therefore a longer biological effect. The end of the ßFSH gene was spliced off and discarded. The carboxy terminal portion (CTP) of ßHCG gene was spliced and introduced to the remaining portion of the ßFSH gene. The new gene was transfected into a Chinese hamster ovary cell line and the resulting chimeric protein FSH-CTP extracted. Animal models have confirmed a longer lasting effect. This model is analogous to recombinant insulin which can now be tailored to the individual patient with combinations of short-, medium- and long-acting preparations. Eventually precise gonadotrophin action will be dissected to the cellular level such that oligopeptides will be created to bind to specific receptors such as in the periovulatory follicle or corpus luteum (Gast, 1995Go).

Conclusion – do we need gonadotrophins?

It could be argued that there is a case to be made for minimizing the use of gonadotrophin preparations – or phasing them out altogether! If we examine in turn the indications for gonadotrophin therapy, we can find an alternative for each.

Ovulation induction for hypogonadotrophic hypogonadism
The most physiological therapy is pulsatile GnRH, which achieves pregnancy rates equivalent to gonadotrophin treatment (Balen et al., 1994Go).

Ovulation induction for polycystic ovary syndrome
Gonadotrophin therapy is used for clomiphene-resistant cases and appears as effective as laparoscopic ovarian diathermy (Gadir et al., 1990Go). Alternative therapies such as insulin-sensitising agents might also have a role to play, as might the in-vitro maturation of immature oocytes (Balen, 1999Go).

Ovulation stimulation for assisted conception
The use of gonadotrophins to stimulate the ovaries has certainly become the mainstay of treatment. The risks of all gonadotrophin treatment regimens includes ovarian hyperstimulation syndrome and the putative risk of ovarian cancer (see review Nugent et al., 1998Go). Improvements in the laboratory might make natural cycle IVF with the collection of one or two oocytes, embryo culture to blastocyst and single blastocyst transfer a real possibility for the future. Continued studies of preimplantation embryos might enable us to identify and select those that are most likely to develop successfully (Edwards and Beard 1999Go). Alternatively we, like others, have been exploring the prospects of the in-vitro maturation of oocytes collected from unstimulated or minimally stimulated ovaries (Wynn et al., 1998Go).

As well as developments in the IVF laboratory and with the gonadotrophin preparations, there have been significant changes in the clinical practice of assisted conception itself. Stimulation protocols are being modified to enhance `friendliness' to the patient (Olivennes and Frydman, 1998Go). Strategies include simple ideas such as using an oral contraceptive agent for 2–3 weeks prior to starting a GnRH agonist; and thereby ensuring pituitary desensitization without the need for additional scans and without the risk of cyst formation (Biljan et al., 1998Go). Ultrasonography has become the mainstay of monitoring both the ovarian and the endometrial response to stimulation, without the need for frequent blood tests of endocrine parameters (Tan, 1994; Wikland et al., 1994Go). The introduction of GnRH antagonists should further improve the welfare of women receiving treatment by first obviating the often distressing side-effects of oestrogen deficency caused by the agonists and second shortening the length of the cycle by being able to commence gonadotrophin therapy in the early follicular phase and preventing an LH surge with appropriately-timed administration of either a single or multiple doses of an antagonist (Olivennes et al., 1998Go; Ganirelix Dose-finding Study Group, 1998Go). The total dose of gonadotrophins used also appears to be reduced when compared with a conventional `long agonist protocol' (Olivennes et al., 1995Go). Furthermore, antagonists can also be used in spontaneous cycles to minimize the cancellation rate of `natural cycle IVF' and thus allow `low burden, user friendly' assisted conception treatment in appropriately selected cases.

Whilst the above treatments are unlikely to entirely replace gonadotrophin stimulation regimens, they should not be discounted, especially if we are to further strive to minimize the risks and costs of assisted reproduction technology. Is it being over-cynical to state that the the majority of the studies that promote the use of gonadotrophins and the various regimens have been supported by the pharmaceutical industry, who have themselves steered away from alternative approaches to fertility therapy?

Gonadotrophin therapy is the mainstay of most forms of fertility therapy and adds appreciably to the cost of the treatment of assisted reproduction therapies – (although it is hoped that the recombinant preparations will be produced in sufficient quantities to enable the prices to be reduced dramatically). Other costs have to be counted in terms of the successful outcome of treatment with a low rate of miscarriage and the birth of healthy, preferably singleton, babies, with no health risks to their mothers. As pertinent now as when first voiced in a debate in this journal, we stated that whilst unifollicular ovulation induction requires a subtle approach, particularly in women with PCOS, superovulation induction is, on the other hand, one of the least subtle of therapeutic procedures as the ovaries are farmed in the quest for eggs (Balen, 1995Go; Edwards et al., 1996Go). Whilst many eggs lead to the chance of many embryos, with the possibility of freezing some for later use, we have seen tremendous advances in the ART laboratory and further study of in-vitro maturation of oocytes, fertilization and implantation might lead us soon to the elective transfer of a single pre-embryo or blastocyst. Still to be addressed is whether it is more healthy for the woman, oocyte and endometrium to have one or two cycles of ovulation simulation for IVF or a few more in which fewer eggs are harvested.

Notes

1 To whom correspondence should be addressed Back

This debate was previously published on Webtrack 58, March 22, 1999

References

Albano, C., Smitz, J., Camus, M. et al. (1996) Pregnancy and childbirth in an in-vitro fertilization cycle after controlled ovarian stimulation in a woman with a history of allergic reaction to human menopausal gonadotrophin. Hum. Reprod., 11, 1632–1633.[Abstract]

Balen, A.H. (1995) Effects of ovulation induction with gonadotrophins on the ovary and uterus and implications for assisted reproduction. Hum. Reprod., 10, 2233–2237.[ISI][Medline]

Balen, A.H. (1999) Polycystic ovary syndrome: mode of treatment. In Shoham, Z., Howles, C.M. and Jacobs, H.S. (eds), Female Infertility Therapy, Curent Practice. Martin Dunitz Ltd, London, UK, pp. 45–68.

Balen, A.H., Tan, S.L. and Jacobs, H.S. (1993) Hypersecretion of luteinising hormone : a significant cause of infertility and miscarriage. Br. J. Obstet. Gynaecol., 100, 1082–1089.[ISI][Medline]

Balen, A.H. and Rose, M. (1994) The control of luteinising hormone secretion in the polycystic ovary syndrome. Cont. Rev. Obstet. Gynaecol., 6, 201–207.

Balen, A.H., Braat, D.D.M., West, C. et al. (1994) Cumulative conception and live birth rates after the treatment of anovulatory infertility. An analysis of of the safety and efficacy of ovulation induction in 200 patients. Hum. Reprod., 9, 1563–1570.[Abstract]

Bergh, C., Howles, C.M., Borg, K. et al. (1997). Recombinant human follicle stimulating hormone (r-hFSH; Gonal-F®): results of a randomized comparative study in women undergoing assisted reproductive techniques. Hum. Reprod., 12, 2133–2139.[Abstract]

Biljan, M.M., Mahutte, N.G., Dean, N. et al. (1998) Effects of pretreatment with an oral contraceptive on the time required to achieve pituitary supppression with GnRH analogues and on subsequent implantation and pregnancy rates. Fertil. Steril., 70, 1063–1069.[ISI][Medline]

Braileanu, G.T., Albanese, C., Card, C. and Chedrese, P.J. (1998) FSH bioactivity in commercial preparations of gonadotropins. Theriogenology, 49, 1031–1037.[ISI][Medline]

Brown, J.B., Evans, J.H., Adey, F.D. et al. (1969) Factors involved in the induction of fertile ovulation with human gonadotrophins. J. Obstet. Gynaecol. Br. Comm., 76, 289–307.[Medline]

Coelingh Bennink, H.J.T., Fauser, B.C.J.M. and Out, H.J. (1998) Recombinant follicle-stimulating hormone (FSH; Puregon) is more efficient than urinary FSH (Metrodin) in women with clomiphene citrate-resistant, normogonadotropic, chronic anovulation: a prospective, mulicenter, assessor-blind, randomized, clinical trial. Fertil. Steril., 69, 19–25.[ISI][Medline]

Daya, S., Gunby, J., Hughes, E.G. et al. (1995) Follicle-stimulating hormone versus human menopausal gonadotropin for in vitro fertilisation cycles: a meta-analysis. Fertil. Steril., 64, 347–354.[ISI][Medline]

Devroey, P., Tournaye, H., van Steirteghem, A. et al. (1998) The use of a 100IU starting dose of recombinant follicle stimulating hormone (Puregon®) in in-vitro fertilisation. Hum. Reprod. 13, 565–566.[Free Full Text]

Donini, P. and Montezemolo, R. (1949) Rassegna di Clinica, Terapia e Scienze Affini. Vol. 48. Biologic Laboratories, Institute Serono. pp. 3–28.

Dore, P.C., Rice, C. and Killick, S. (1994) Human gonadotrophin preparations may cause allergic reaction. Br. Med. J., 308, 1509–1510.[Free Full Text]

Edwards, R.G., Lobo, R. and Bouchard, P. (1996) Time to revolutionize ovarian stimulation. Hum. Reprod., 11, 917–919.[ISI][Medline]

Edwards, R.G. and Beard, H.K. (1999) Blastocyst stage transfer: pitfalls and benefits. Is the success of human IVF more a matter of genetics and evolution than growing blastocysts? Hum. Reprod., 14, 1–4.[Free Full Text]

European Recombinant Human LH Study Group (1998) Recombinant human LH to support recombinant human FSH-induced follicular development in LH- and FSH-deficient anovulatory women: A dose finding study. J. Clin. Endocrinol. Metab., 83, 1507–1514.[Abstract/Free Full Text]

Fares, F.A., Suganuma, N., Nishimori, K. et al. (1992) Design of a long-acting follitropin agonist by fusing the C-terminal sequence of the chorionic gonadotropin beta subunit to the follitropin beta subunit. Proc. Natl. Acad. Sci. USA, 89, 4304–8.[Abstract]

Fauser, B.C., Donderwinkel, and Schoot, D.C. (1993) The step-down principle in gonadotrophin treatment and the role of GnRH analogues. Ballières Clin. Obstet. Gynaecol., 7, 309–330.

Fulghesu, A.M., Lanzone, A., Gida, C. et al. (1992) Ovulation induction with human menopausal gonadotrophin versus follicle stimulating hormone after pituitary suppression by gonadotrophin-releasing hormone agonist in polycystic ovary disease: A cross over study. J. Reprod Med., 37, 834–840.[ISI][Medline]

Gadir, A.A., Mowafi, R.S., Alnaser, H.M.I. et al. (1990) Ovarian electrocautery versus human menopausal gonadotrophins and pure follicle stimulating hormone therapy in the treatment of patients with polycystic ovarian disease. Clin. Endocrinol. (Oxf.), 33, 585–592.[ISI][Medline]

Ganirelix Dose-finding Study Group (1998) A double-blind, randomised dose-finding study to assess the efficacy of the GnRH antagonist Ganirelix (Org 37462) to prevent premature LH surges in women undergoing ovaraian stimulation with recombinant FSH (Puregon). Hum. Reprod., 13, 3023–3031.[Abstract]

Gast, M.J. (1995) Evolution of clinical agents for ovulation induction. Am J. Obstet Gynecol., 172, 753–759.[ISI][Medline]

Hamilton-Fairley, D., Kiddy, D.S., Watson, H. et al. (1991) Low-dose gonadotrophin therapy for induction of ovulation in 100 women with polycystic ovary syndrome. Hum. Reprod., 6, 1095–1099.[Abstract]

Hayden, C.J., Rutherford, A.J. and Balen, A.H. (1999a) Induction of ovulation using a starting dose of 50 units of recombinant human follicle stimulating hormone (Puregon) Fertil. Steril., in press.

Hayden, C.A., Balen, A.H. and Rutherford, A.J. (1999b) Recombinant gonadotrophins. Br. J. Obstet. Gynaecol., in press.

Homburg, R., Eshel, A., Kilborn, J. et al. (1990) Combined luteinising hormone releasing hormone analogue and exogenous gonadotrophins for the treatment of infertility associated with polycystic ovaries. Hum. Reprod., 5, 32–35.[Abstract]

Howles, C.M., Barri, P., Cittadini, E. et al. (1992) Metrodin HP Clinical experiences with a new highly purified follicle stimulating hormone preparation suitable for subcutaneous administration. In Lunenfeld, B. (ed.), FSH Alone in Ovulation Induction. Parthenon Publishing Group, New York, USA, pp. 45–61.

Howles, C.M., Loumaye, E., Giroud, D. et al (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. Hum. Reprod., 9, 424–430.[Abstract]

Jacob, S., Drudy, L., Conroy, R. and Harrison, R.F. (1998) Outcome from consecutive in-vitro fertilisation/intracytoplasmic sperm injection attempts in the final group treated with urinary gonadotrophins and the first group treated with recombinant follicle stimulating hormone. Hum. Reprod., 13, 1783–1787.[Abstract]

Jacobs, H.S., Porter, R., Eshel, A. and Craft, I. (1987) Profertility uses of luteinising hormone releasing hormone agonist anologues. In Vickery, B.H. and Nestor, J.J. (eds), LHRH and its Analogs. MTP Press Ltd, Lancaster, UK, pp. 303–322.

Jansen, C.A.M. and Van Os, H.C. (1996) Puregon without analogues: an oxymoron. Gynaecol. Endocrinol. 10 (Suppl. 1), 34.

Kousta, E., White, D.M., Piazzi, A. et al. (1996) Successful induction of ovulation and completed pregnancy using recombinant human luteinizing hormone and follicle-stimulating hormone in a woman with Kallman's syndrome Hum. Reprod., 11, 70–71.

Lambert, A., Rodgers, M., Mitchell, R. et al. (1995) In-vitro biopotency and glycoform distribution of recombinant human follicle stimulating hormone (Org 32489), Metrodin and Metrodin-HP. Mol. Hum. Reprod., 1, see Hum Reprod., 10, 1928–1935.[Abstract]

Lambert, A., Talbot, J.A., Anobile, C.J. and Robertson, W.R. (1998) Gonadotrophin heterogeneity and biopotency: implications for assisted reproduction. Mol. Hum Reprod., 4, 619–629.[Abstract]

Le Cotonnec, Porchet, H., Beltrami, V. et al. (1994a) Clinical pharmacology of recombinant human follicle-stimulating hormone (FSH). i. Comparative pharmacokinetics with urinary human FSH. Fertil. Steril., 61, 669–678.[ISI][Medline]

Le Cotonnec, Porchet, H., Beltrami, V. et al. (1994b) Clinical pharmacology of recombinant human follicle-stimulating hormone (FSH). ii. Single doses and steady state pharmacokinetics. Fertil. Steril., 61, 679–686.[ISI][Medline]

Loumaye, E., Campbell, R. and Salat-Baroux, J. (1995) Human follicle stimulating hormone produced by recombinant DNA technology: a review for clinicians. Hum. Reprod. Update, 1, 188–199.[ISI]

Loumaye, E., Martineau, I., Piazzi, A. et al. (1996) Clinical assessment of human gonadotrophins produced by recombinant DNA technology. Hum. Reprod., 11, 95–107.[Abstract]

Lunenfeld, B. and Insler, V. (1974) Classifiaction of amenorrhoeic states and their treatment by ovulation induction. Clin. Endocrinol., 3, 223–237.[ISI][Medline]

McDonough, P.G. (1995) Recombinant follicle stimulating hormone and luteinizing hormone – mimicking nature and beyond. Fertil. Steril., 64, 211–212.[ISI][Medline]

MacDougall, M.J., Tan, S.L., Balen, A.H. and Jacobs, H.S. (1993) A controlled study comparing patients with and without polycystic ovaries undergoing in-vitro fertilization. Hum. Reprod., 8, 233–237.[Abstract]

Meniru, G.I. (1999) Puregon a `good' or `super' drug?! Hum. Reprod., 14, 1409–1411.[Free Full Text]

Nugent, D., Salha, O., Balen, A.H. and Rutherford, A.J. (1998) Ovarian neoplasia and subfertility treatments. Br. J. Obstet. Gynaecol., 105, 584–591.[ISI][Medline]

Olivennes, F., Fanchin, R., Bouchard, P. et al (1995) Scheduled administration of GnRH antagonist (Cetrorelix) on day 8 of IVF cycles: a pilot study. Hum. Reprod., 10, 1382–1386.[Abstract]

Olivennes, F., Alvarez, S., Bouchard, P. et al. (1998) The use of a GnRH anatgonist (Cetrorelix) in a single dose protocol in IVF-embryo transfer: a dose finding study of 3 versus 2 mg. Hum. Reprod., 13, 2411–2414.[Abstract]

Olivennes, F. and Frydman, R. (1998) Friendly IVF: The way of the future? Hum. Reprod., 13, 1121–1124.[Free Full Text]

Out, H.J., Reimitz, P.E., Coelingh Bennink, H.J.T/European Puregon Collaborative IVF study group (1995). A prospective, randomised, multicentre study comparing recombinant FSH (Puregon ) either given intramuscularly or subcutaneously in subjects undergoing IVF. Hum. Reprod., 10 (Abstr. Book 1), 6.

Out, H.J., Mannaerts, B.M.J.L., Driessen, S.G.A.J. and Coelingh Benninck, H.J.T. (1996) Recombinant follicle stimulating hormone (rFSH; Puregon) in assisted reproduction: More oocytes, more pregnancies. Results from five comparative studies. Hum. Reprod. Update, 2, 162–171.[Abstract/Free Full Text]

Out, H.J., Mannerts, B.M.J.L., Driessen, S.G.A.J. and Coelingh Benninck, H.J.T. (1995) A prospective randomised assessor-blind, multicentre study comparing recombinant and urinary follicle stimulating hormone (Puregon vs. Metrodin) in in-vitro fertilisation. Hum. Reprod., 10, 2534–2540.[Abstract]

Recombinant Human FSH Study Group (1995) Clinical assessment of recombinant human follicle-stimulating hormone in stimulating ovarian follicular development before in vitro fertilsation. Fertil. Steril., 63, 77–86.[ISI][Medline]

Rose, M.P., Gaines Das, R.E. and Balen, A.H. (1999) Definition and measurement of FSH. Endocr. Rev., in press.

Sagle, M.A., Hamilton-Fairley, D., Kiddy, D. and Franks, S. (1991) A comparative, randomised study of low-dose human menopausal gonadotrophin and FSH in women with polycystic ovary syndrome. Fertil. Steril., 55, 56–60.[ISI][Medline]

Schoot, D.C., Coelingh Benninck, H.J.T., Mannaerts, B.M.J.L. et al. (1992) Human recombinant follicle-stimulating hormone induces growth of preovulatory follicles without concommitant increase in androgen and estrogen biosynthesis in a woman with isolated gonadotrophin deficiency. J. Clin. Endocrinol. Metab., 74, 1471–1473.[Abstract]

Schramm, R.D. and Bavister, B.D. (1994) Follicle-stimulating hormone priming of rhesus monkeys enhances meiotic and developmental competence of oocytes matured in vitro. Biol. Reprod., 51, 904–912.[Abstract]

Shoham, Z., Patel, A. and Jacobs, H.S. (1991a) Polycystic ovary syndrome: safety and effectiveness of stepwise and low-dose administration of purified follicle stimulating hormone. Fertil. Steril., 55, 1051–1056.[ISI][Medline]

Shoham, Z., Balen, A.H., Patel, A. and Jacobs, H.S. (1991b) Results of ovulation induction using human menopausal gonadotropin or purified follicle-stimulating hormone in hypogonadotropic hypogonadism patients. Fertil. Steril., 56, 1048–1053.[ISI][Medline]

Staessen, C., Janssenswillen, C., Van Den Abbeel, E. et al. (1993) Avoidance of triplet pregnancies by elective transfer of two good quality embryos. Hum. Reprod., 8, 1650–1653.[Abstract]

van Weissenbruch, M.M, Schoemaker, H.C, Drexhage H.A. and Shoemaker, J. (1993) Pharmacodynamics of human menopausal gonadotrophin (HMG) and follicle stimulating hormone (FSH). The importance of the FSH concentration in initiating follicular growth in polycystic ovary-like disease Hum. Reprod., 8, 813–821.[Abstract]

Venturoli, S., Orsini, L.F., Paradisi, R. et al. (1986) Human urinary FSH and hMG in induction of multiple follicle growth and ovulation. Fertil. Steril., 45, 30–35.[ISI][Medline]

Wang, C.F. and Gemzell, C. (1980) The use of human gonadotrophins for induction of ovulation in women with polycystic ovarian disease. Fertil. Steril., 33, 479–486.[ISI][Medline]

Wikland M., Borg, J., Hamberger, L. et al. (1994) Simplification of IVF: minimal monitoring and the use of subcutaneous highly purified FSH administration for ovulation induction. Hum. Reprod., 9, 1430–1436.[Abstract]

Wynn, P., Picton, H.M., Krapez, J.A. et al. (1998) Pretreatment with FSH promotes the numbers of human oocytes reaching metaphase II by in-vitro maturation. Hum. Reprod., 13, 3132–3138.[Abstract]