Reviews Induction of follicular growth and ovulation with urinary and recombinant gonadotrophins*

Transcription

1 RBMOnline - Vol 3. No Reproductive BioMedicine Online webpaper 2001/050 on web 16 July 2001 Reviews Induction of follicular growth and ovulation with urinary and recombinant gonadotrophins* Dr Francisco Rísquez Francisco Rísquez was born in From he was Assistant in the In Vitro Fertilization Centre Baudelocque (under Prof. Jean-René Zorn and Lise Cedard), Paris, France, where he also taught assisted fertilization techniques to visiting French and foreign physicians. Between he was infertility specialist at the Centro Médico Docente La Trinidad and Instituto Médico la Floresta, Caracas, Venezuela and teaching coordinator of Gynecology and Ultrasound. From he was Chief of the Infertility Clinic, Centro Médico Docente La Trinidad, Caracas, Venezuela and in 2000 became Director there. He is well known as a pioneer in transcervical tubal cannulation and microendoscopic techniques, such as microlaparoscopy, and lectures widely on reproductive medicine (expertise on gynaecological endocrinology IVF, Fallopian tubes and endoscopy) as well as writing many scientific articles and books. He has been the recipient of rewards from the Venezuelan Society of Surgery (1985) and Valencia (1992). Francisco Rísquez Centro Médico Docente La Trinidad Correspondence: Av. Luis Roche, Edif. Altamira Palace, Apt. 11-B, Caracas, Venezuela. risquezf@cantv.net Abstract Gonadotrophins have been used since the 1930s. By the 1990s, a pure biological extract of FSH was obtained. The increase in the demand for new assisted reproduction techniques led to the creation of recombinant FSH, 99% pure and highly consistent between batches, with no LH activity, and a high specific activity. In gonadotrophin protocols, follicular growth can be monitored with regular ultrasounds and/or hormonal testing, mainly oestradiol in the follicular phase. In assisted reproduction the adoption of ovulation induction strategies with GnRH analogues, control the endogenous secretion of LH, avoiding cancellations due to an early LH surge, and premature ovulation. Depending on the moment when pituitary suppression starts, there are short, ultra-short and long protocols The use of long protocols not only increases pregnancies and live births, it also allows an easier programming and simplifies IVF treatments. Ovarian stimulation entails some risks such as ovarian hyperstimulation syndrome and multiple pregnancies that can be avoided in many cases with an accurate prediction and an active prevention. Keywords: gonadotrophin, monofollicular and multifollicular protocols, ovarian hyperstimulation syndrome, recombinant FSH Introduction The use of gonadotrophins for ovarian stimulation has become essential for many forms of assisted human conception. Pituitary extracts and urinary human preparation were characertistic of the last half of the twenthieth century. Moreover, until recently, the induction of ovulation with pharmacological agents including anti-oestrogens was restricted mainly to women with ovulation alterations or anovulatory infertility, particularly those with oligomenorrhoea or amenorrhoea. Anovulatory infertility is due to a lack of ovulation. In this condition, the follicle does not rupture and the oocyte does not emerge. Unfortunately, at present there is no method to verify fully the ovulatory process and the only absolute test of ovulation is pregnancy. Anti-oestrogens such as clomiphene have been used effectively to treat women who presumably have a defect in the luteal phase, as well as to induce the development of multiple follicles *Chapter taken from a forthcoming book in Spanish on Modern Assisted Conception edited by RG Edwards and F Risquez. Supported by Serono. to facilitate the collection of multiple oocytes to be used in assisted reproduction techniques, and even as an empirical treatment for women who have unexplained infertility. The introduction of in-vitro fertilization (IVF), greatly increased the need for gonadotrophin stimulation of follicle growth and ovulation. The first pregnancy obtained by Robert Edwards and Patrick Steptoe was ectopic following stimulation with human menopausal gonadotrophin (HMG) and human chorionic gonadotrophin (HCG) and transfer of a single blastocyst. Soon after the first baby, Louise Brown, was born using assisted fertilization following a natural cycle and obtained by the same team of pioneers (Steptoe and Edwards, 1978), and there was enough evidence to show that the possibilities for IVF increase significantly when more than one embryo is transferred. Ever since then, there has been an important development in different stimulation protocols (Edwards et al., 1980). 54

2 Today, some authors state that the future of gonadotrophins for assisted reproduction is in the recently developed recombinant products, which include FSH, LH and HCG. The present trend in ovulation induction strategies is to simplify them as much as possible, making its administration easier, more affordable, and more convenient for couples, perhaps with lower doses of rfsh, and formulae that are more friendly. The recent use of GnRH antagonists produces regimes that are simpler and shorter, with lower doses and immediate reversal. The use of short or long acting rfsh molecules could help individualize protocols and therefore reduce the cost of the treatment. The use of rfsh implies an improvement in ovarian stimulation, which is very beneficial to the patient. The identification of the clinical characteristics of patients with anovulation that do not respond to clomiphene allows the early use of gonadotrophin therapy, and it has more benefits, economic as well as for their health. Moreover, if the response to the ovulation induction can be predicted for each patient, then it would be possible to design individual regimes that reduce the risk of ovarian stimulation and multiple pregnancy. The stimulation of ovulation using gonadotrophins is still essential for the management of anovulatory infertility. These hormones are also used in cases such as hypothalamichypophysial insufficiency, hypothalamic dysfunction, hyperandrogenism, cervical iatrogenic factor, failure with clomiphene citrate, as well as in assisted reproduction cycles. Different gonadotrophin preparations History Gonadotrophins have been used since 1930, when they were extracted from the pituitary gland of pigs or from the serum of mares. Animal extracts were used for ~30 years. During the 1950s, researchers started obtaining gonadotrophins from the pituitary gland of cadavers or from the urine of menopausal women. Gonadotrophins from the pituitary are not used any more given that they may cause diseases, such as Jacob Creutzfeld disease observed in patients treated with these pituitary extracts as a hormonal growth supplement. The process to extract the HMG was designed in 1947 by Pietro Domini, a chemist from Serono Laboratories in Rome. These preparations have a low specific activity and at present specific activity 100 IU/FSH mg only 1 2% of the proteins is gonadotrophin. The preparations have an equal amount of follicle stimulant hormone (FSH) and luteinizing hormone (LH), (~75 IU of each in every ampoule). Since the beginning, HMG proved its efficiency and thus was established as the regular treatment. In the 1980s, new formulae were developed, mainly with FSH activity (75 IU) and a lower proportion of LH activity (<0.7 IU), a low specific activity at between FSH/mg of the protein (u-fsh), Metrodin, Serono Laboratories). The LH was removed through immunochromatography. This product has been used alone or with HMG in the treatment of polycystic ovaries, as well as in protocols for assisted reproduction. By 1993, a highly purified urine-derived FSH was introduced (u-fsh HP, Metrodin HP), with a specific activity of ~9000 IU FSH/mg of protein. Metrodin HP is the first pure biological extract that can be characterized with physico-chemical tests, and offers an increase in consistency between the different bands. Given its high purity, it can be injected subcutaneously, contrary to the previous preparations that must be administered intramuscularly. Pure FSH has proven to be as effective as HMG to stimulate follicular development. This shows that the residual endogenous luteinizing hormone (LH), or the LH contained in existing preparations is enough to support follicular development, despite its low quantity (Bentick et al., 1988; Edelstein et al., 1990). Despite the high specific activity of Metrodin HP, the initial material is heterogeneous, and requires the collection of millions of litres of urine from thousands of donors. Since the preparations differ depending on their origin, their activity also varies considerably. It is accordingly very difficult to prepare protocols with consistent formulae to induce ovulation, and they need to be varied according to the need of each patient (Figure 1). 55 Figure 1. The evolution of follicle stimulating hormone preparations (modified from Howles and Wikland, 1999).

3 In recent years, an increase in the use of assisted reproduction techniques, as well as difficulties in covering the demand through urine collection, created problems in the production process. This led to the use of recombinant DNA technology to create recombinant follicle stimulating hormone (FSH) (Howles and Wikland, 1999). In this article, the use of gonadotrophins is emphasized, particularly those emerging from state of the art technology, including recombinant FSH, in monofollicular as well as multifollicular stimulation protocols. The article also mentions possible adverse effects and how to avoid them. Human menopausal gonadotrophins (HMG) and recombinant FSH (rfsh) Until recently, the only preparations available were HMG and pure FSH. Since they were obtained from urine extracts, with a purity of only 1 2% and highly contaminated with proteins, they could cause local reactions in the tissue and pain, requiring a deep intramuscular injection. These preparations have a reduced biological activity and their variable isoforms of gonadotrophins with different pharmakinetic properties are responsible for the wide variations between individuals in the half-life of these hormones. In general, the half-life of pure FSH after intramuscular administration varies between 30 and 40 h, whereas LH has a shorter half-life. For this reason, a long duration HCG is administered as a substitute for the LH surge. Recombinant FSH (rfsh) is obtained from Chinese hamsters ovary cells (CHO), known to be host cells in the production of recombinant glycoproteins. CHO cells can express FSH activity biologically, in sufficient amounts to make the production process viable. A genomic clone that contains the complete sequence of the FSHb subunit, alone or together with the gene of the a subunit, is transferred to the CHO cells. The polypeptide chain of rfsh is identical to the natural one, as opposed to the carbohydrated natural structures that can be identical or closely related. Like natural FSH preparations, rfsh has a considerable heterogeneity, and its bioactivity has been confirmed in in-vitro bioassays and in-vivo clinical studies. Moreover, chimeric molecules have been synthesized with a longer half-life, by modifying the carboxy-peptide end, and are available for phase I clinical tests. The specific bioactivity of rfsh is IU FSH/mg of proteins (Loumaye and Howles, 1999; Table 1). The high degree of purity allows the subcutaneous administration (SC) of rfsh (Table 1). The advantages of rfsh, compared to the urine-derived product can be summarized as an increase in consistency of the final product, high biological purity, which allows its SC injection by the patient herself, as well as its chemical characterization for better quality control, very low concentrations of oxidized or degraded FSH forms, complete lack of LH so allowing studies on the control of ovarian follicular function, and the production of rfsh molecules with short or long activity, allowing a better induction of ovulation (Devroey et al., 1994; Jansen et al., 1998; Tarlatzis and Billi, 1998; Daya and Gunby, 1999). Theoretical and clinical bases for induction with gonadotrophins Treatment of anovulatory groups I and II of the World Health Organization A large number of anovulatory patients who are infertile have an insufficient response to antioestrogenic medication, so gonadotrophins represent an effective secondary treatment. According to the classification of the World Health Organization (WHO), these patients are either on group I, characterized by a low oestrogen production related to hypogonadotrophic hypogonadism, or in group II, with persistent oestrogen secretion. In both groups, the clinical challenge is to obtain egg maturity with a minimum number of follicles (preferably one) to obtain a simple pregnancy without invoking ovarian hyperstimulation syndrome (OHSS). Although it seems that this can be achieved through simple manipulation of gonadotrophins, more than with the selection of the molecule itself, the recent introduction of rfsh justifies an evaluation of this subject. In fact, differences in the carbohydrates of glycoproteins can produce differences in metabolism and biological activity. Moreover, the change in isoform distribution offers an approach to modulate the biological biopotency. In 1995, the first preparation of rfsh was registered in the European Community (Gonal-F, Ares-Serono, Geneva, Switzerland), and the second became available in 1996 (Puregon, NV Organon, Oss, Holland). So today there are two preparations of recombinant gonadotrophins. Both are identical with respect to the peptide but in their sequence there are small differences in isoform profile. Some recent clinical data show that both preparations, administered in equal doses based on an equivalent international unit, produce a stimulation with identical characteristics whilst others show differences. Nevertheless, when Puregon is administered SC, there is evidence of a lower tolerance (Howles and Wikland, 1999). Further studies are required to evaluate fully the extent of these Table 1. Characteristics of different human gonadotrophin preparations (from Loumaye and Howles, 1999) Preparation Year Source of Protein concen- FSH bioassay LH bioassay Physico- FSH mass Admin. registered FSH tration (IU/mg) (IU) (IU) chemical QC content control route Meno 1/ urine > none no im Meno 2/ urine > none no im Urofolli 1982 urine > <0.1 none no im Urofolli HP 1992 urine ~ < no sc Folli 1995 CHO ~ yes sc Meno = menotropin; Urofolli = urofollitropin; Folli = follitropin; CHO = Chinese hamster ovary cells; FSH = follicle stimulating hormone; LH = luteinizing hormone; QC = quality conrol; HP = highly purified; im = intramuscular; sc = subcutaneous. 56

4 differences. The use of rfsh has been approved for two main conditions: anovulation in patients resistant to clomiphene citrate, belonging to group II of the WHO, and for the multifollicular ovulation stimulation of normo-ovulatory women undergoing assisted reproduction cycles (Table 2). The hypothesis of a synergism of gonadotrophins, introduced more than half a century ago (Fevold, 1941), emphasizes the importance of an interplay between FSH and LH for normal oestrogen production and follicular development. Later, this concept was supported by studies where pure FSH was injected to immature mice causing an increase in the weight of the ovary without affecting the growth of the uterus. This suggests that FSH alone is enough to promote follicular development without a concomitant production of oestrogen. Likewise, the administration of pituitary or pure FSH to hypogonadotrophic women leads to follicular development without the secretion of oestrogens (Schoot et al., 1992). These observations support the two cells, two gonadotrophins theory in follicular steroidogenesis (Figure 2; Short, 1962). According to this hypothesis, the cells of the ovarian theca stimulated by LH secrete androgens that pass through the basal membrane within the granulosa cells, where an FSH induced enzymatic system converts androgens into oestrogens (Figure 2). Table 2. Results of ovarian stimulation in 28 hypogonadotropic hypogonadal women treated with 150 IU/day rfsh (Gonal-F) and different doses of rlh (from Loumaye et al., 1995). rlh (IU/day) Patients (n) Pre-ovulatory 250 ± ± ± ± 423 oestradiol (pmol/l) Endometrial 4.3 ± ± ± ± 0.9 thickness (mm) Pre-ovulatory prog- <3 <3 <3 <3 esterone (mmol/l) Midluteal prog- 50 ± ± ± ± 24 esterone(mmol/l) Pregnancies (n) Patients with group I hypogonadotrophic hypogonadism (WHO) can be treated with rfsh. The causes for anovulation are rare in anovulatory group I (WHO), since there is a central failure in the secretion of gonadotrophins which translates into abnormally low serum concentrations of gonadotrophins and almost no secretion of oestrogen from the ovary. In the first case (Schoot et al., 1992), multifollicular growth was observed through ultrasound, but the concentrations of oestradiol remained very low. Moreover, the concentrations of oestradiol in the follicular fluid were ~1500 times lower than expected in the normal pre-ovulatory stage, and serum progesterone did not increase after the administration of HCG. These observations were confirmed in other studies with a large number of patients. The first observations were hence confirmed, indicating that it is not necessary to have high concentrations of intrafollicular oestradiol for the maturation of follicles in humans. Another interesting observation was that immunoreactive inhibin increased a few days before a small rise in serum oestradiol. Thus, there is some speculation that the inhibin produced by the granulosa, stimulated by FSH, may act as a paracrine regulator to stimulate the production of androgens in the thecal cells. In turn, these cells provide a base for the synthesis of oestradiol from the cells in the granulosa. The fact that serum progesterone does not increase after injection of HCG may be explained by the low local concentration of oestradiol, which is insufficient for the induction of LH receptors. Finally, plasma androgen concentration remains stable during stimulation, showing that rfsh does not have an intrinsic LH activity (Shoham et al, 1993). The two-cell theory of ovarian steroidogenesis and the role of FSH All these data confirm the dissociation between the mitogenic and steroidogenic activity of FSH, and emphasise that although follicular development is predominantly FSH dependent, steroidogenesis is supported mainly on the synergy between FSH and LH (Fevold, 1941; Short, 1962). This last concept was recently evaluated in anovulatory patients from group I (WHO). A group of patients was treated with fixed doses of rfsh (Gonal-F, Serono Switzerland;150 IU/day) and increasing doses of r-lh (Luveris Serono Switzerland; IU/day). The high doses of r-lh could promote an optimum secretion of oestradiol and improve the luteinizing capacity of the follicle after the administration of HCG (Loumaye et al., 1995). 57 Figure 2. The two-cell theory of ovarian steroidogenesis. Other preliminary studies performed in woman exposed to rfsh preparation, such as hypogonadotrophic-hypogonadic women, show that there is an extremely low concentration of oestradiol in the follicular fluid and the plasma, even though there is multifollicular development (Schoot et al., 1994). Therefore, LH is required in very small amounts to induce an adequate androgen production in the theca cells, which will then be converted to oestrogens under the influence of FSH. Moreover, humans may not require oestrogens for follicular maturation, as opposed to the evidence found in rodents. Oestrogen seems to be necessary to prepare the genital tract for the implantation of the embryo. rfsh is therefore effective to stimulate the follicle in anovulatory patients from group I (WHO), but it must be

5 administered together with LH to obtain an adequate follicular steroidogenesis, as shown by oestradiol secretion, endometrial growth and progesterone secretion in the luteal phase. The second indication that needs to be evaluated is the stimulation of a single follicle in anovulatory group II (WHO). The first cases reported were women whose anovulation was related to the polycystic ovary syndrome, resistant to clomiphene citrate. In both studies, low dose protocols were used since they are safe and effective for patients with high sensitivity to gonadotrophins. The administration of rfsh resulted in an exponential increase in serum oestradiol and in immunoreactive inhibin, showing that exposure to endogenous LH is enough to maintain the follicular development induced by FSH. These data support the hypothesis of synergism between gonadotrophins in oestrogen production ( two-cell theory ). Relative clinical efficiency of u-fsh and rfsh The pregnancies and births resulting from this therapy confirmed even further the mechanism of normal follicular development. Furthermore, other authors have used pure FSH or rfsh to induce ovulation in group II (WHO), with no difference in the efficiency of both protocols. Data obtained from ovulation inductions for assisted reproduction techniques indicate that rfsh is more efficient than u-fsh, since it produces higher concentrations of oestradiol, and more follicles, recovered oocytes and embryos. This research also shows a greater efficiency using a significantly lower total dose in a shorter treatment period (Jansen et al., 1998). Multicentre studies have also shown a greater efficiency of rfsh compared to u-fsh for chronic anovulatory patients who are normo-gonadotrophic, i.e. classified as group II (WHO), showing that rfsh produces more follicles and a higher concentration of plasma oestrogens. These differences can be explained by the composition of the new rfsh molecule. Nevertheless, there was an increase in cancellations due to ovarian hyperstimulation, so it is advisable to start with low doses (37.5 IU FSH) and increase them carefully. This was not found with Gonal-F. The clinical usefulness of rfsh combined with GnRH analogues was proven in 1992, with the first pregnancies obtained by ovulatory women in assisted reproduction cycles (IVF) (Germond et al., 1992; Devroey et al., 1992). These first pregnancies in women undergoing IVF using rfsh were reported by two separate groups using the two existing preparations (Gonal-F and Puregón; Table 3). The stimulation of follicles was successful, and the collection of oocytes that were later fertilized was sufficiently good so as to transfer the embryos and achieve these first pregnancies. On the other hand, a pilot study examined the efficiency of five different protocols using GnRH analogues and rfsh for follicular induction and oocyte development in IVF. In all the groups treated with rfsh, there was follicular growth and an increase in oestradiol and inhibin concentrations, but LH concentrations remained low in women treated with triptorelin. Results show that GnRH analogues and rfsh are effective and even though there is a profound suppression of LH, sufficient residual LH sustains normal steroidogenesis. Later prospective multicentre studies performed with rfsh versus pure FSH, showed that rfsh is more effective than pure FSH to induce ovulation for IVF (Bergh et al., 1997). There was also a significant increase in recovered oocytes and a better quality of embryos in the group using rfsh. A lower number of rfsh ampoules was used to stimulate ovaries and in a shorter period of time as compared to pure FSH, which indicates that rfsh is more efficient than u-fsh. Although both the number of follicles and serum oestradiol concentration are higher during the day of HCG administration, the incidence of OHSS is higher but not statistically significant. Therefore, taking into account the higher potency of rfsh preparations, it is essential to monitor it carefully, to avoid OHSS. The results of this study showed that rfsh is more effective than urinederived FSH to induce multifollicular development and obtain pregnancies. A prospective, randomized study showed that rfsh is more effective than urine-derived FSH to induce multifollicular development in couples undergoing intracytoplasmic sperm injection (ICSI). The ovarian response rate was lower using urine-derived FSH, leading to the conclusion that there must be less variability between the bands in recombinant gonadotrophins. Recently, another multicentre prospective study evaluated the efficiency of fixed rfsh doses in women suppressed by GnRH analogue (Healy and Out, 1998). The group using 100 IU rfsh produced fewer oocytes and embryos than the group using 200 Table 3. Recombinant versus highly purified urinary FSH in women undergoing assisted reproduction treatment: characteristics of patients receiving HCG ( values are means ± SD) (from Bergh et al., 1997) rfsh ufsh HP P-value Ampoules (75 IU) FSH 21.9 ± ± 13.4 < FSH (days) 11.0 ± ± 3.7 <0.001 Oocytes retrieved 12.2 ± ± 4.4 < Oestradiol on day of HCG (nmol/l) 6.55 ± ± 3.9 <0.001 Embryos cleaved on day ± ± 3.5 < Embryos transferred 2.0 ± ± 0.4 NS Pregnancy/cycle (%) 53/119 (45) 42/114 (37) NS Twin pregnancies (%) 17 (32) 9 (25) NS Implantation rate (%) NS NS = not significant 58

6 Figure 3. FSH threshold and window: monofollicular (left panel) and multifollicular (right panel). 59 IU. There was an increase in the cancellation rate in the group with 100 IU and a higher incidence of OHSS with 200 IU. Also the miscarriage rate was significantly higher with the 100 IU dose these studies together demonstrate that 150 IU per day is probably an adequate starting dose in most patients. Another recent double-blind, randomized study compared rfsh with pure FSH in a group of women undergoing assisted reproduction, and concluded that rfsh is more efficient than pure FSH to induce multiple follicular development. OHSS can be associated within the use of urinary preparations. It is not significantly higher with rfsh if the right dose is used. Experience with rfsh is rapidly increasing (Howles, 2000). Even though there is some ovarian hyperstimulation, it is directly related to the pharmacological properties of the compound. rfsh is well tolerated when injected SC or intramuscularly. SC administration allows self administration by the patient. In some cases there has been severe systemic reactions to u-fsh mediated by E immunoglobulins and presumably related to non-gonadotrophic proteins. This does not happen when rfsh is administered. It is important to note that no anti-rfsh antibody has been detected in women treated with rfsh preparations (Figure 3). Monofollicular induction and monofollicular ovulation The number of follicles recruited and matured in a natural or induced cycle depends on the number of follicles stimulated by FSH, the duration of the stimulation, and the sensitivity of the ovary. These factors are explained by the concepts of FSH threshold and FSH window, proposed for follicular growth in vivo, at the end of the 1980s. The objective of monofollicular ovulation induction is to stimulate the development of a good quality oocyte, with high probabilities of becoming a good quality embryo, a simple pregnancy and a healthy child. It is clear that this is a different objective from the goals of IVF, where protocols are designed to promote multifollicular development which requires higher doses of FSH. Women who have polycystic ovaries (PCO) and fail to respond to clomiphene citrate might ovulate when stimulated with gonadotrophins. The objective of ovulation induction with gonadotrophins, as with clomiphene citrate, is the formation of a single dominant follicle (Homburg and Howles, 1999). In spontaneous cycles this is achieved in the beginning of the cycle, with a transient increase in plasma FSH concentrations, above threshold values. These concentrations decrease later, preventing the development of more than one follicle. To achieve the development of a single follicle using exogenous gonadotrophins, it is necessary to give a specific treatment and a use a good monitoring protocol. Although several ovulation induction methods have been described using gonadotrophins, the two most frequently found in the literature and in clinical practice are the low-dose step-up protocol, and more recently the low-dose step-down protocol. The dose of administered gonadotrophins depends on the response obtained (Macklon et al., 1999). Stimulatory regimens Step-up protocol The concept of a FSH threshold was proposed by Brown in 1978 and is becoming increasingly accepted. It is based on clinical observations of cycles stimulated with gonadotrophins and emphasizes the narrow margin in FSH requirements to begin follicular stepping-up growth (Brown, 1978). It suggests stepping-up the administration of gonadotrophins in a typical protocol or conventional step-up until there is sufficient ovarian response as established by ultrasound and hormone tests. Later, other authors suggested a slower increase in gonadotrophin doses to reduce even further the incidence of ovarian hyperstimulation. Today, this continuous regime using the lowdose step-up protocol is widely used to induce ovulation in oligomenorrhoeic infertile women. A recent comparative study between the continuous regime with a low dose protocol, versus the conventional protocol using rfsh in anovulatory infertile women from group II (WHO) shows that rfsh is safe and effective in both cases; it also confirms that the low-dose

7 FSH LEVELS FSH LEVELS protocol is associated with a higher mono or bifollicular development (Hedon et al., 1998). Initial doses of gonadotrophins, and the nature of incremental doses as the cycle proceeds have been calculated by various investigators (van Santbrink et al., 1995). From these studies it can be concluded that the greater apparent effectiveness of rfsh justifies its preferred use in ovulation stimulation procedures, using the low-dose protocol for patients in group II (WHO). The initial doses must be selected according to the clinical characteristics of the patients, and after a careful examination of previous ovarian stimulations. In the case of monofollicular ovarian stimulations, the threshold dose varies for each patient, and the step-up protocols increase the doses systematically and in small increments. Nevertheless, the interval and the amount of the initial dose has been determined in the practice, and it depends on the type and quality of the exogenous gonadotrophins used. The conventional step-up protocol uses an initial FSH dose of IU/day increasing by 75 IU after 5 7 days (Macklon et al., 1999). However, it has many complications. Up to 36% of multiple pregnancies have been reported and ovarian hyperstimulation can occur in up to 14% of the treated cycles. Therefore, many centres have abandoned this protocol, and now prefer the chronic low-dose step-up protocol, which was designed to reach the FSH threshold gradually, minimizing an excessive stimulation and therefore the risk of developing multiple preovulatory follicles (Figure 4). In this protocol, the initial SC or intramuscular dose of FSH is IU/day; the dose is increased only if after 14 days a response is not observed as assesed by ultrasound and serum oestradiol concentrations. Increments of 37.5 IU are administered in weekly intervals up to a maximum of 225 IU/day. The detection of an ovarian response is an indication to continue with the present dose until HCG can be given to induce ovulation. Multiple folliculogenesis and hyperstimulation are less common than in the conventional protocol, and pregnancy rates are similar. Step-down protocol DAYS OF GONADOTROPHIN Figure 4. Continuous low-dose step-up protocol (adapted from Macklon et al., 1999). Sequential protocols using u-fsh have been described, where an initial continuous low dose administration is followed by a step-down regime after follicular selection has occurred. This method has proven safe and effective for patients with DAYS OF GONADOTROPHIN Figure 5. Continuous low-dose step-down protocol (adapted from Macklon et al., 1999). polycystic ovaries. Other studies have used rfsh. Since various combinations of urinary and recombinant hormone preparations have been utilised, with and without the combined use of GnRH agonists, many studies have been applied to this treatment. Step-down protocols for ovulation induction are more similar to the physiology of the normal cycle (Figure 5). The theory of the FSH window emphasizes the importance of decreasing plasma FSH concentrations for the development of a single dominant follicle. Therapy with 150 IU FSH/day begins soon after spontaneous or progesterone induced bleeding, and it continues until a dominant follicle (>10 mm) is observed through a vaginal ultrasound. The dose is decreased to IU/day, followed by another reduction 3 days later to 75 IU/day, which is continued until HCG is administered to induce ovulation (Macklon et al., 1999). The appropriate initial dose can be calculated using a low-dose step-up protocol for the first treatment cycle. The low-dose step-up protocol is safe and effective, but it is longer and more expensive. Initial experience with the step-down protocol suggests that the duration of the treatment and the total amount of gonadotrophin decrease, as compared to the low-dose step-up protocol, provides better follicular growth more frequently (van Santbrink, 1995). These findings were recently confirmed, comparing both protocols in a prospective and randomized study. It was shown that there was a greater incidence of monofollicular development in women undergoing ovulation induction with the step-down protocol, thus reducing the risk of multiple pregnancy and hyperstimulation. However, the safety and efficiency of the step-down protocol must be confirmed by large multicentre studies. The ovarian response to gonadotrophin therapy is monitored using transvaginal ultrasound to measure follicular diameter. The test is done every 2 or 4 days and must be focused to identify mid-sized follicles. Between 5000 and IU HCG is administered intramuscularly on the day that there is at least one follicle >18 mm diameter. If there are more than three follicles >16 mm, stimulation must be suspended, HCG must not be administered, and a barrier contraceptive must be used to prevent multiple pregnancies and ovarian hyperstimulation. Plasma oestradiol measurement can also be useful, and preovulatory concentrations above normal average values (14 40 ng/dl or pmol/l) might predict an impending hyperstimulation. However, it is not clear how much 60

8 information oestradiol concentrations add to information already obtained with transvaginal ultrasound. There are various other adjunct therapies. These include suppression with dexamethasone from suprarenal androgens, with agonists to suppress high LH concentrations, with dopaminergic agonists when there is hyperprolactinaemia, and with growth hormone. Nevertheless, none of these additional therapies has increased the number of pregnancies in randomized prospective studies. Alternating regimes: low doses of rfsh in alternating days Alternate day regimen using pure FSH was advocated over 10 years ago (McFaul et al., 1989). In those studies the effectiveness of intramuscular FSH administered as daily or alternate-day injections to patients with polycystic ovarian disease (PCOD) who previously failed to ovulate on clomiphene citrate was compared. It was concluded that alternate-day FSH therapy is as effective as daily FSH in achieving ovulation and pregnancies in patients with PCOD resistant to treatment with clomiphene citrate. Moreover, there were no differences in any of the parameters measured between daily and alternate-day FSH. The fact that today there are pure hormone preparations such as rfsh has led to more stringent and reliable investigations about the mode of administration and the dose of FSH in assisted reproduction programmes. Recently, the possibility of administering rfsh less frequently and in a lower dose has been investigated for ovulation induction programmes. The concept takes into consideration the increased biopotency of rfsh compared to u-fsh, and knowledge about the pharmacokinetics of rfsh, with its half-life of h. Alternating day regimes have been proposed for monofollicle development in anovulatory patients resistant to clomiphene citrate (Figure 6). The higher frequency of simple pregnancies and lack of ovarian hyperstimulation cases with low-dose stepup protocols show how matured oocytes have a high quality and how these protocols help to mature follicles richer in FSH receptors. A preliminary study (Buckler et al., 1999) found that FSH concentrations, measured h after administration of alternate doses of rfsh (100 or 50 IU) in similar patients, did not rise above the basal endogenous concentrations. Perhaps there is a relative reduction in FSH concentration, which must occur 24 h before injection of the next dose, leads to atresia of follicles with few FSH receptors. This increases the possibility of monofollicular development and a decrease in overstimulation in patients with a tendency to develop OHSS. The values of assays detecting oestradiol and inhibin A increases during the administration of gonadotrophin, are is in line with the concept that inhibin A is a marker of the maturity of the dominant follicle. Moreover, studies show that concentrations of inhibin A and B during treatment were similar to those reported in the follicular phase of the normal menstrual cycle. In other words, this protocol is similar to the normal menstrual cycle regarding time and hormonal parameters. The role of gonadotrophins in polycystic ovary syndrome is not clear (Buckler et al., 1999). An abnormal secretion of gonadotrophins, including an increase in basal LH and in the LH:FSH ratio has been reported (Conway et al., 1989). It has been suggested that the high frequency in the secretion of GnRH can play an important role in the lack of gonadotrophins in patients with polycystic ovarian syndrome (PCOS). A recent study shows that infertile anovulatory patients with PCOS who do not respond to endogenous FSH, could respond quickly in <2 weeks in most cases to produce monofollicular ovulation with low doses of rfsh undetectable in blood above endogenous FSH. A possible interpretation is that small follicles in patients with PCOS respond to a blend of rfsh glycoforms, and that the glycoforms of endogenous FSH recruits them, but does not promote either the growth or the development of the dominant follicle. Unfortunately, little is known about the quality of endogenous FSH glycoforms in PCOS, although it is known that LH is markedly less acidic. At present, research is being conducted to study the possibility that a blend of FSH glycoforms predominates in PCOS and is responsible for recruitment. Using rfsh in low doses and in alternating days can induce a physiological and biochemical medium similar to that found in the normal menstrual cycle. Therefore, it is necessary to focus more on the physiology of the ovary to develop strategies for new treatments. However, this approach has to be validated. 61 Figure 6. Low dose alternate day rfsh protocol. Figure 7. Clomiphene citrate (CC) and gonadotrophins protocol (NET: norethisterone).

9 Further studies are required to evaluate fully the differences in results obtained, between pure FSH (McFaul et al., 1990) and rfsh for alternate regimens. Multifollicular induction (ovulation induction) in assisted reproduction Starting with the first pregnancy obtained with IVF, which was ectopic, and the first live birth in 1978 from a natural cycle, there has been a particular interest in ovulation induction. Based initially on studies in mice, the use of HMG and HCG was found to be highly effective in the 1970s, although numbers of growing follicles and mature oocytes varied erratically between patients (Edwards et al., 1980). In the 1980s, Steptoe and Edwards, Trounson, and Jones continued developing IVF techniques, including the use of gonadotrophins to stimulate ovulation (Edwards et al., 1980; Trounson et al., 1981; Jones, 1985). Since then, there have been many changes in the development of gonadotrophins, and in the protocols for multifollicular induction. The objective of multifollicular induction for IVF is to produce more eggs to obtain a sufficient amount of embryos and select those with good quality to be transferred. Evidently, if the patient responds well to ovulation induction, and produces high quality oocytes, the chances for success is greater than if she does not. Early use of ovulation induction in IVF cycles indicated that success was determined mainly by the number of transferred embryos. It has been defined that the replacement or transfer of three or more healthy embryos optimizes the possibility of obtaining a pregnancy. However, it was also shown that the replacement of three embryos or more raises the possibility of multiple pregnancies, without a higher rate of pregnancies. With good sperm preparation, and four or five good quality eggs, there is a higher chance of having three embryos with normal morphology. Therefore, the natural cycle was replaced by ovulation induction, although in the early 90s there has been a return to this natural procedure, demonstrating its value for a limited group of patients. The monitoring of ovulation induction is based on following the growth of the follicles through ultrasound, clinical examinations of the patient, and laboratory hormone tests, usually oestrogen concentrations. In general, the higher the oestradiol concentration, the more follicles are developing. When the physician considers that follicular development has reached an appropriate concentration to obtain enough eggs, based on laboratory tests (oestrogen concentrations), ultrasound and physical examinations, the hormone HCG is given to trigger ovulation. Oocytes are collected h after administration of HCG. It is necessary to monitor the growth and development of follicles, and avoid the possibility of OHSS. Ultrasound must be performed frequently and in series to follow up the finer details of follicular growth in the ovary. Ultrasound must be used at the beginning of the cycle and especially from cycle day 8 until the time of oocyte collection. Oocyte collection can also be done with the help of ultrasound. It is considered minor surgery and can be performed under local anaesthesia. The vaginal transducer is placed in the vagina and an aspiration needle is aligned with the vaginal ultrasound transducer, aimed directly at the ovary. The ultrasound image allows the physician to guide the needle to each follicle and aspirate the follicular contents. The procedure is well accepted by physicians and has few side-effects. Oocyte aspiration aided by ultrasound is performed on an outpatient basis and ensures a good collection of follicular contents. Clomiphene citrate and gonadotrophins This protocol is based on the negative feedback that oestrogens produce on endogenous gonadotrophins. Clomiphene citrate acts on the hypothalamus and the pituitary, producing a moderate hypersecretion of pituitary gonadotrophins. If it is administered soon after the beginning of menstruation, clomiphene citrate can stimulate the growth of small follicles, and endogenous gonadotrophins or the administration of exogenous gonadotrophins, can maintain that growth and the recruitment of the follicular cohort. This regime was widely accepted in the beginning of fertilization techniques, but it was difficult to predict the spontaneous LH surge (Figure 7). A premature LH surge leads to a high rate of cancellations, which may be due to spontaneous ovulation before oocyte collection, or to the recovery of post-mature ovules with a reduced fertility (Trounson and Wood, 1984; Jones, 1985; Steptoe et al., 1986). Given that the LH surge cannot be predicted with the simple observation of follicular size, with the absolute concentration of oestrogens, or with the oestrogen growth rate, most successes have been obtained with a careful monitoring of endogenous LH secretion during the late follicular phase. When the LH surge is detected, HCG is administered, and the moment to collect the eggs is carefully calculated, an acceptable rate of success can be obtained. Several investigators still believe this treatment is preferable to agonists and FSH since it is more gentle and much less expensive. There is a relationship between oocyte quality and LH in the late follicular phase. Fertilization failure due to bad oocyte quality is correlated with high LH concentrations in the late follicular phase. There is more evidence of the negative effects of high LH concentrations on ovarian function in patients cancelled before obtaining the oocytes, due to a bad follicular development with high LH concentrations. The important element in these findings is that high LH concentrations in the follicular phase are incompatible with implantation and predict an early loss of pregnancies after ovulation induction, IVF, or natural conception. The physiopathological base for high LH concentrations in the late follicular phase can be related in part to the individual, but it seems to be more related to the response to clomiphene citrate administration. Clomiphene citrate is administered from cycle days 2 to 6 inclusive. HMG or FSH is administered at a dose of 150 IU/day (two ampoules) from cycle day 5 up to the day of administration of HCG. Changes can be done to this basic regime, taking into consideration the response to previous stimulations. The dose of clomiphene citrate can be reduced to 50 mg. Pure FSH or rfsh can be added from cycle day 2. The daily dose of gonadotrophin can vary according to the response. The administration of gonadotrophins can be omitted on the day of HCG administration. Due to the great possibility of a premature LH surge, it is 62

10 63 essential to monitor it carefully and continuously, with endocrinological profiles as well as ultrasounds measure follicles during the late follicular phase. It is advisable to use the ultrasound daily and to do endocrinological tests on cycle day 9. Daily estimates of plasma LH, oestradiol and progesterone concentrations are necessary. Once a follicle >14 mm is obtained, urine LH concentrations can be measured every 4 h. Once the beginning of the LH surge is detected, it must be sustained with HCG (5000 or IU), and the collection of oocytes should be planned at 36 h after the onset of the surge or h after LH concentrations double for the first time. The criteria for HCG administration for ovulation induction in clomiphene citrate and gonadotrophin cycles is the spontaneous beginning of the LH surge, or one or more follicles with an average diameter of mm. Other authors administer it when they are ~17 mm. The greater the size of the follicle, the greater the possibility of triggering endogenous LH when HCG is administered. A simple dose of IU HCG is administered to induce ovulation, and the collection of oocytes occurs 34.5 h later. Programming is difficult in these cycles due to the spontaneous LH surge, but some planning can be done in many cases. The best way is to work backwards, from the day of the anticipated oocyte collection, and programme the onset of menstruation to occur 12 days before. This is achieved through the extension of the preceding luteal phase with the administration of noritestosterone (30 mg/day, in divided doses) or another type of previous therapy. The results of clomiphene citrate and gonadotrophin stimulation show a pregnancy rate per transfer of ~25% or more in different centres, which is still lower in comparison with cycles using GnRH agonists (Macnamee et al., 1989). Gonadotrophin-releasing hormone (GnRH) agonists and gonadotrophins for IVF GnRH agonists have been used since the late 1980 s to the present, and many studies using Depot analogues were initially performed demonstrating their usefulness not only for assisted reproduction, but also for the alleviation of endometriosis and uterine myomas (Zorn et al., 1990). The main objective of GnRH analogues prevent the spontaneous discharge or secretion of LH from triggering ovulation; they also allow more follicular recruitment, thus increasing the number of ovules and embryos. The suppression of the pituitary with GnRH agonists reduces the endogenous secretion of LH, and this increases fertilization and pregnancy rates. The first exposure to GnRH agonists induces a massive secretion of pituitary gonadotrophins. Another consequence is a decrease in the number of GnRH receptors, as well as a reduction in sensitivity to endogenous GnRH. The constant occupancy of the newly synthesized receptors maintains a low sensitivity, even though the receptors for gonadotrophin storage are restored. This results in a drop in circulating LH concentrations, and a slight reduction in circulating FSH. Once the administration of GnRH analogues is suspended, normal pituitary function is restored only when the synthesis of new receptors occurs. As gonadotrophins become sensitive to endogenous GnRH, the concentrations of LH and FSH are restored. Ovulation induction strategies, combining the use of GnRH agonists and HMG or rfsh fall into the following categories: ultra-short, short, long and ultra-long protocols. Depending on the category, the administration of GnRH agonists can occur prior to the end of the secretory phase or the luteal phase of the preceding cycle. This is called long protocol. GnRH agonists could be given during the initial stage or follicular phase of the treatment cycle, in the so-called short protocol. If the long protocol extends in time, it is called ultra-long, and if the short protocol utilises only a few doses of the analogue at the beginning of the follicular phase, it is called ultra-short protocol. Nowadays there is a large variety of GnRH analogues. It is difficult to compare them, because the different studies present not only different GnRH analogues, but also different administration methods. However, it is clearly proven that suppression is more profound with the depot administration than with the daily subcutaneous administration, even though the latter has the advantage of dose adjustment during the administration interval. Nasal administration has a reduced absorption rate, compared to subcutaneous and intravenous administration but is still highly effective. Concerns were expressed about the inadvertent exposure of the early conceptus to GnRH analogues, which may occur in 1% of the cases when they are used in the middle of the previous luteal phase (long protocol). Later studies suggest that these pregnancies are not a mere coincidence, but rather they are related to the positive effect of analogues in fertility through the action of LH on the corpus luteum. There are no increases in the number of abortions, ectopic pregnancies or malformations in patients exposed to these products. Short and ultra-short protocols In the short and ultra-short protocols, the initial stimulatory phase of GnRH agonists is used to stimulate the recruitment of follicles and to maintain the hyposecretion of endogenous gonadotrophins in the later part of the follicular phase (Figure 8). In both protocols, the initial secretory phase of GnRH agonists is used for follicular recruitment and early development. To take advantage of the secretory phase, in both regimes the GnRH agonist is administered early, for example, around cycle day 2, and exceptionally on cycle day 3. In the case of the ultra-short protocol, GnRH agonist administration begins on cycle day 1 for 3 days (Macnamee et al., 1989), whereas in the short protocol, the administration continues until the administration of HCG. It is necessary to ensure the absence of the corpus luteum through endocrinological analysis and ultrasound to prevent its rescue with the administration of GnRH agonists. To enhance follicular development, gonadotrophins are administered as the pituitary is suppressed. Anomalies in endogenous LH activity and LH surges are not frequent in these regimes (<1% of the cases), but are generally associated to high concentrations of oestradiol in patients with multicystic or polycystic ovaries. In the case of the ultra-short regime, pituitary sensitivity is recovered approximately 9 to 14 days

11 Figure 8. Multifollicular protocols utilising GnRH agonists (agnrh) and gonadotrophins: short and ultra-short protocols. E2 = oestradiol. after the last dose of the GnRH analogue. For a woman who has normal ovulation, the criteria for HCG administration to induce ovulation is reached before pituitary sensitivity is recovered. This does not happen in poor responders, so it is advisable to perform blood and urine tests if the treatment exceeds 12 days. Long and ultra-long protocols The strategies of the long and ultra-long GnRH protocols are based on the desensitization of the pituitary for a longer period of time, in order to induce a temporary state of hypogonadotrophic hypogonadism (Figure 9). Follicular recruitment and growth is thus stimulated with exogenous FSH. In other words, a state of hypogonadotrophic hypogonadism is produced which lasts between 8 and 21 days. Once the desensitization occurs, the dose of GnRH agonists can be reduced to maintenance concentrations, preventing an LH surge, and reducing but not eliminating basal LH secretion. In normal ovulatory women, desensitization can be achieved after 7 days of administration of GnRH agonists, but in oligomenorrhoeic women, particularly those with PCOS, it may take more time. It is advisable to confirm pituitary suppression, through endocrinological tests as well as with ultrasound. Endocrinologically, LH must be <5 IU/l, oestradiol <5 pg/ml, and progesterone <1 ng/ml. Ovulation induction can be obtained with the daily administration of gonadotrophins, once the suppression occurs. It is advisable to begin using agonists in the middle of the luteal phase of the preceding cycle, i.e days before the onset of menstruation. This method has the advantage of a decrease in the tone of LH, due to progesterone activity. It also avoids the consequences of starting the administration of analogues in the early stages of the follicular phase, such as the recovery of the corpus luteum, alterations in normal folliculogenesis, expansion of cyst structures within the ovary, and stimulation of the recruitment of growing follicles, due to the initial secretion of agonists (Macnamee and Brinsden, 1999). There are two types of long protocol. The long follicular protocol, based on the administration of GnRH analogues from cycle day 1, for ~12 to 14 days, whereas the long luteal protocol is based on the administration of GnRH from the middle of the luteal phase of the previous cycle. The follicular regime has the Figure 9. Multifollicular protocols utilising GnRH agonists (agnrh) and gonadotrophins: long protocol. E2 = oestradiol. advantage that it does not require the confirmation of follicular phase, and it is preferred for oligo-ovulatory patients. Nevertheless, some of these patients receiving daily treatment with GnRH agonists have an intense response, with stimulation of follicular cysts and persistently high concentrations of oestradiol. Recently, an ultra-long protocol was proposed by some groups in the case of women with endometriosis for IVF cycles, stating that it produces a higher pregnancy rate (Marcus and Edwards, 1994). During 4 months, GnRH analogues are administered followed by gonadotrophins as in the previous protocols. However, other groups do not corroborate the increase in pregnancy rates. The total number of ampoules is higher than with others protocols. GnRH analogues and gonadotrophins, their monitoring, criteria for ovulation induction and programming In short protocols, the subcutaneous administration of 150 IU/day of rfsh begins 1 or 2 days after the administration of the agonist, when gonadotrophin concentrations start to decline. In long protocols, rfsh is started subcutaneously at 150 IU/day, after a base ultrasound, and when LH, oestradiol and progesterone concentrations indicate that suppression has been achieved through the use of agonists. The initial dose of rfsh in short and in long protocols may vary between 150 and 450 IU/day, given subcutaneously, depending on the age of the patient, the presence of polycystic ovaries, or a history of poor response. In general, once ovarian stimulation has begun, regardless of the protocol used, monitoring is performed as described previously, through ultrasound and hormone testing (oestradiol). The determining element, particularly in long protocols, is the evaluation of pituitary suppression. Even though ultrasound can evaluate the degree of activity of the ovary, only the circulating concentrations of LH, oestradiol and progesterone can confirm suppression before starting the administration of gonadotrophins. As in the other ovulation induction regimes, the daily dose must be adjusted according to the response. Once gonadotrophins start the growth of the follicles, monitoring can be done only through ultrasound. Nevertheless, establishing the 64

12 Figure 10. Pregnancy rates using different GnRH agonist protocols (from Daya, 1997). oestradiol concentrations in the final part of the follicular phase is very valuable. Since endogenous LH secretion is abolished in these regimes, it is rare for early luteinization to occur, and it does occur close to the time when the HCG is indicated. The main criterion for HCG administration to induce ovulation is when a reasonable number of follicles has been detected. Two follicles must be dominant at 18 mm diameter. There is great flexibility in the time of HCG administration. Some physicians administer it only when the dominant follicle is 20 or 22 mm diameter, depending on oestradiol concentration and size of the other follicles. HCG is indicated at a dose of IU, and oocyte collection is done h later, usually through vaginal ultrasound. The use of agonists enables the oocyte collection procedure to be well planned (Zorn et al., 1987; Rísquez et al., 1990). Short protocols can be programmed successfully, just by planning the onset of menstruation through an extension of the preceding luteal phase. In long protocols, once ovarian suppression is obtained, oocyte collection can be achieved by delaying the administration of gonadotrophins. In normal ovulatory women, 10 days of gonadotrophin stimulation is enough to reach the criteria for ovulation induction, and ensures sufficient oestrogenic preparation of the endometrium. Therefore, stimulation must begin 12 days before the day established for oocyte collection. In long protocols, starting the administration of analogues in the luteal phase is more practical, gives more flexibility in the planning of IVF cycles, and simplifies the therapy. protocol increases the probabilities of conception and live births after IVF (Figure 10; Daya, 1997). Several factors may explain the superiority of the long protocol. The initial action of GnRH analogues in short protocols is to increase LH concentration. This can lead to a stimulation of the corpus luteum ( rescue ) and an increase in androgens from the theca, which can harm folliculogenesis. The degree of LH suppression is variable with the short protocol, and this has been mentioned as the reason why 5 10% of the cycles can be complicated with a premature LH surge. The exposure of the developing follicle to high LH concentrations can adversely affect the reproductive process and this is particularly evident in patients in whom the return to basal LH concentrations takes longer than normal, for example, in polycystic ovaries or in cases where cysts are created as a result of the administration of GnRH agonists. It must be understood that the administration of GnRH agonists required to maintain pituitary suppression decreases throughout the treatment. Minimum ovarian stimulation Despite the methods used nowadays with their intense ovulatory induction, there are still attempts to increase pregnancy rates with the spontaneous or natural menstrual cycle. Even though it is more natural and less invasive, pregnancy rates are lower. There is still a very high rate of cancellations, and the appearance of an LH surge causes logistic and programming difficulties (Fauser et al., 1999). The recent introduction of GnRH antagonists that competitively block GnRH receptors, decreasing gonadotrophin concentrations very rapidly and therefore preventing a premature LH surge, gives a new opportunity to revise strategies and re-introduce minimum ovarian stimulation for IVF (Figure 11; Olivennes and Frydman, 1998; Rongieres- Bertrand et al., 1999; de Jong, Macklon and Fauser, 2000). The role of the GnRH antagonists in ovulation induction and assisted reproduction has been discussed (Chillik and Acosta, On the one hand, the use of minimum ovarian stimulation would have several advantages. It is more comfortable for the patient, less complex, uses shorter regimes, has less probability of complications among them multiple pregnancies and OHSS, 65 Even though the use of the different protocols is well understood, the selection of the protocol to be used is more complex and has led to several comparative studies. Large, well-controlled studies have shown that there is an increase in the take-home baby rate using protocols with GnRH agonists, compared to stimulation with clomiphene citrate and gonadotrophins. This greater success is due to an increase in the number and quality of oocytes, achieved through a reduction in LH concentrations. The advantage of short and ultra-short protocols is the short duration of the stimulation, with fewer ampoules of gonadotrophins and at a lower cost. However, after long studies comparing the results of the long protocol and the short/ultrashort protocols, it has been clearly demonstrated that the long Figure 11. GnRH antagonist and gonadotrophins. E2 = oestradiol.

13 and is less expensive. On the other hand it has the following disadvantages: fewer oocytes are obtained for fertilization in vitro, IVF could not be easily programmed, there would be fewer embryos for cryopreservation and more attempts would be needed. Moreover, there are still some unanswered questions which should be addressed, including birth rates, effects on oocyte quality and fertilization rates, on embryo quality, on endometrial receptivity, and requirements to support the luteal phase. Less complex ovarian stimulation protocols, together with a better selection of embryos and uterine receptivity could lead to a significant advance in IVF (Olivennes et al., 2000). Poor responders: ovarian ageing and evaluation of the functional reserve of the ovary. Occasionally, patients display a poor response. When such responses are repeated, both in frequency and in duration, then the patient can be classified as a poor responder. For practical purposes, a patient who has undergone ovularian stimulation for assisted reproduction and in two cycles has no response or an altered response is classified as a poor responder. Several factors affect ovarian response, including age, family history of premature ovarian insufficiency, previous surgery, endometriosis, obesity, idiopathic infertility, and smoking. Nevertheless, age is probably the primary determining factor for the success of fertility treatments, particularly IVF. Age is directly related to ovarian ageing, which becomes noticeable in the late thirties age group (Figure 12). Perimenopausal transition as a natural phenomenon varies in each woman and is directly related to a progressive loss of follicular content. There is a reduction in inhibin concentrations, and the pituitary reacts by increasing FSH secretion, leading to an increase in basal oestradiol concentrations (Muttukrishna et al., 2000). In some cases there is a greater risk of excessive LH surge and ovulation, and others display early luteinization. An acceleration in follicular growth shortens the follicular phase and therefore the cycle itself. The granulosa cells may have a decreased function, as indicated by reduced production of steroids and glycoproteins in vitro, decrease in mitosis, and increase of apoptosis. In aged ovaries there is an hypoxic environment in the follicles, and there are fewer granulosa cells (Seifer et al., 1996). Patients who are apparently normal but who have a poor ovulatory response show an imbalance in the ovary pituitary hypothalamus relationship. Presumably, the cause of this imbalance is a functional antagonism between gonadotrophin-release factors (GnRH) and inhibiting factors (gonadotrophin surge inhibitory factor (GNSIF)). Nevertheless, it must be noted that if an older patient has a good ovarian reserve and her ovaries respond well, the possibilities of getting pregnant are similar to those of a younger patient, provided she has good quality embryos. Other parameters are available to evaluate the functional reserve of the ovary and to give a prognosis. Among them, basal FSH must be measured during the early follicular phase (day 3 5). According to some authors, basal FSH concentrations >15 miu/ml are related to a poor stimulation response (Barri et al., 1998). If basal concentrations of LH are <3mIU on day 3, it is related to a bad prognosis. For some authors a basal oestradiol concentration >80pb/ml has a discriminatory value only in those patients who respond poorly to stimulation, even though they have normal FSH values. The clomiphene challenge test (Devoto and Chile, personal communication) evaluates the way in which the ovary controls FSH secretion (Loumaye et al., 1990; Hofmann et al., 1996). A possible explanation for FSH sensitivity to clomiphene citrate is the low secretion of inhibin in ovarian follicles with functional deficiencies. This test is very useful in older patients who have been subjected to ovarian surgery or have endometriosis. Other practical or potential parameters to evaluate the functional reserve of the ovary include assays of inhibin (Lockwood et al., 1998), dynamic tests, and ultrasound. Dynamic tests with leuprolide are not considered necessary, since the information they provide is given by basal hormonal concentrations. Ultrasound with tridimensional ecographs has shown that the number of follicles (between 2 5 mm) in a young patient with poor response, and normal basal FSH concentrations are low when compared to a control group. Figure 12. Age (arbitrary scale) and the number of follicles (arbitrary scale). Therefore, older patients, and young patients having abnormal endocrinological profiles may have a poor response. Alternative protocols for diagnosis and treatment are used in young patients with a normal endocrinological profile who respond poorly to stimulation. Several alternative protocols have been used in poor responders. Among them, reducing the dose of GnRH agonist, changing it, modifying the moment of its administration, using growth hormone, changing gonadotrophin dosage, using clomiphene citrate and urine-derived gonadotrophins, and using bromocryptine produce variable responses. In many cases no improvement whatsoever is obtained. It is still too soon to evaluate the potential of recombinant hormones for these cases. Nevertheless, natural cycles and alternative protocols must be used before egg donation, which is the last hope for these patients. 66

14 67 Table 4. Classification of ovarian hyperstimulation syndrome (OHSS) Clinical and biological Mild Moderate Severe profiles Ovaries <5 cm <12 cm >12 cm Abdominal pain Ovarian cysts Abdominal distension + + Nausea +/- + Vomiting +/- + Diarrhoea +/- + Ascites + Hydrothorax +/- Haemoconcentration + Coagulation abnormalities + Risks of ovarian stimulation: ovarian hyperstimulation syndrome (OHSS) Characteristics The main risks of ovarian stimulation are multiple pregnancies (Chillik and Acosta, 2001), and OHSS. A good understanding of the problem, its prediction and prevention can reduce and prevent a difficult clinical situation. The syndrome is an iatrogenic complication of ovulation induction. The cause of the syndrome is not completely clear. The main aetiological factor is angiotensin II, which increases vascular permeability, and causes vasoconstriction and neovascular proliferation; consequently fluid exudes from the intravascular to the extravascular compartment which could lead to fluid accumulation in the abdominal cavity (ascites), a reduction of the intravascular volume, and haemoconcentration (Rizk et al, 1997). The condition was originally described in the 1960s, after using ovulation induction techniques in anovulatory women. The syndrome also occurs after ovarian stimulation for assisted reproduction procedures. Contrary to what is desired in ovulation induction in anovulatory women, the aim of ovarian stimulation for assisted reproduction is to produce multiple large follicles which are aspirated to collect several oocytes, thus avoiding the consequences of hyperstimulation at the moment of embryo transference. There are several classifications regarding the degrees of ovarian stimulation (Table 4), but no consensus has been reached, which complicates any comparison between the different studies. In general, three OHSS categories are accepted: mild hyperstimulation, where there is an increase in the size of the ovary with the presence of small cysts; moderate hyperstimulation, also with ovarian cysts, abdominal distention, pelvic pain, possibly with nausea, vomits and/or diarrhoea; and severe hyperstimulation, which involves large ovarian cysts, ascites and/or hydrothorax, possibly a marked haemoconcentration with an increase in blood viscosity, which may result in coagulation anomalies and a decrease in renal perfusion. Recently, another classification was proposed and has only two categories: moderate hyperstimulation, which includes the symptoms of the mild and moderate cases of the previous classification, and severe hyperstimulation, which is basically the same as in the previous classification. In the cases of ovulation induction for IVF procedures, a mild or moderate ovarian hyperstimulation is relatively common, but not the severe cases, which occur less frequently in ovarian stimulations (Rizk and Aboulghar, 1999). For practical purposes even though there are several classifications of the severity of ovarian stimulation those patients with complex symptoms can be divided into severe and critical, which require hospitalization and intensive care. The severe cases can include ascites with or without hydrothorax (fluid accumulation in the pleural cavity) an increase in haematocrites and leukocytes, decreased urination (oliguria), an increase in creatinine concentrations, hepatic dysfunction, and anasarca (generalized oedema). In the critical hyperstimulation syndrome, the conditions described for the severe case are worse and there may be either renal insufficiency, thromboplebitis or respiratory distress syndrome (Balasch et al., 1990). Several factors affect the incidence of OHSS (Table 5). One relates to the selection of patients. There is a higher incidence in group II of the WHO, particularly those with PCOS. Depending on the different ovulation induction protocols used, there is a greater propensity to OHSS with GnRH analogues. It is also observed with the use of HMG, and pure FSH is not safer than HMG. The use of rfsh does not prevent OHSS, its effects being comparable to pure FSH, even using conventional low dose protocols. Clomiphene citrate rarely produces a severe OHSS. Rare cases of ovarian hyperstimulation are reported in spontaneous cycles, and during spontaneous pregnancies. Surgery is not recommended in these cases, but nevertheless it is performed due to mistaken diagnosis. The greatest risk factor for OHSS is the administration of exogenous HCG, particularly in the luteal phase, or the endogenous HCG that appears during pregnancy. Therefore, there are two types of syndrome: an early syndrome, which appears 3 7 days after the administration of HCG and is related to oestradiol concentrations as well as the number of aspirated oocytes; and the late hyperstimulation syndrome which emerges days after the injection of HCG with a severity related to the number of gestational sacs. The quality and maturity of the oocytes is reduced in OHSS, which decreases the fertilization rate. It has no effect on the quality of transferred embryos or the pregnancy rate. The effect on oocyte quality may be due to the prevalence of polycystic ovaries in this group of patients. Management The most effective treatment could be an accurate prediction and an active prevention (Table 6). Care is needed with young patients who have PCOS or who have had a previous follicular hyperstimulation syndrome. A combination of ultrasound and endocrinological monitoring of follicular development has been suggested as a means for prediction during ovarian stimulation. Regarding endocrinological monitoring, the best predictive element of OHSS is plasma oestradiol concentrations; one study found that below 1000 pg/ml, there were no cases of OHSS and these authors considered that above

15 Table 5. Incidence of OHSS in HMG/HCG cycles (from Rizk and Aboulghar, 1999) Author Year Mild (%) Moderate (%) Severe (%) Rabau et al Tyler Taymor et al Thompson and Hansen Jewelewicz et al Hammond Caspi et al Lunenfeld and Insler Schwartz et al Table 6. Prediction and prevention of OHSS Prediction Young Polycystic ovarian syndrome Previous OHSS Ultrasound High serum oestradiol Prevention Coasting Lower gonadotrophins Freezing embryos for later use Progesterone for luteal support Others: follicular aspiration, albumin suggested (Asch et al., 1993), since its many actions include the sequestering of vaso-active substances secreted by the corpus luteum, and others produced additionally as a result of OHSS. Albumin has a half-life of days, and the patient generally develops the symptoms 3 10 days after the injection of HCG. Nevertheless, several authors have recently reported cases of severe OHSS even with the prophylactic administration of intravenous albumin (Mukherjee et al., 1995) pg/ml HCG should not be administered because hyperstimulation occurred in all patients (Haning et al., 1983). Nevertheless, several authors have reported severe OHSS even with oestradiol concentrations <1500 pg/ml. On the other hand, only a small number of patients with an excessive oestrogen concentration can develop severe OHSS (Schwartz et al., 1980). The possibility of predicting OHSS increases with follicular monitoring through ultrasound, preferably transvaginal, which allows the number, size and distribution pattern of follicles to be taken into condideration. The basal volume of the ovary, measured through ultrasound before ovarian stimulation may be a predictor, and colour Doppler is useful in this respect. Some authors suggest that intra-ovarian vascular resistance in patients undergoing ovulation induction could be a predictor in patients with a particular risk of developing OHSS. It is generally accepted that the combination of plasma oestradiol and ultrasound is the best predictor of OHSS. In practice, when high concentrations of oestradiol are detected indicating a possibility of OHSS, the gonadotrophin dose must be suspended. Treatment with the agonist is continued and the patient is monitored for 3 or 4 days. When oestradiol concentrations decline to pg/ml, HCG can be administered. This delay in giving HCG is known as coasting and its application reduces the possibility of developing severe OHSS (Aboulghar et al., 2000). Other techniques have been used to prevent this syndrome, including the prolonged use of GnRH agonists, avoiding the administration of HCG, using lower dosages of gonadotrophins and at less frequent intervals, decreasing the dose of HCG or eliminating it completely, not administering HCG during the luteal phase and avoiding endogenous HCG, freezing embryos for later use and finally administering progesterone in the luteal phase. The administration of intravenous albumin has also been Others suggest the use of GnRH agonists to trigger ovulation. Evidently, this method cannot be used in patients who were prescribed protocols that include GnRH analogues. Multiple follicular aspirations have also been used prior to HCG administration, but this has been criticised by other authors because there is no evidence of improvement. Recently, some authors have found a significant reduction of the incidence of OHSS by using the LHRH antagonist cetrorelix (Cetrotide) in ovarian stimulation for assisted reproduction (Ludwig et al., 2000). The treatment for the moderate syndrome is to rest in bed and to increase the intake of fluids containing sodium chloride and other electrolytes. In the severe case without an abnormal biochemical profile, the patient is treated with aspirations of the ascitic fluid and intravenous fluid therapy in an outpatient basis. In cases of severe hyperstimulation with hydroelectrolytic imbalance or other complications, the patient must be hospitalized. There are recent studies on a system of continuous self-transfusion with a venous peritoneal derivation for severe cases. During hospitalization, it is necessary to perform frequent monitoring and to control the most frequent complications, including vascular factors, with coagulation anomalies and haemoconcentration, the cerebrovascular complication being the most serious of all. These factors also include hepatic dysfunction; respiratory complications and respiratory distress as a result of ascites; renal complications caused by hypovolaemia secondary to the exudation of fluid from the intravascular to the extravascular space and gastrointestinal complications which must be carefully monitored because gastro-intestinal symptoms are one of the first manifestations of OHSS. Surgical treatment is reserved only for those cases where annex cysts rupture or twist, or where ectopic pregnancy occurs. If the situation becomes critical, and if the patient is pregnant, the pregnancy must be terminated. 68

16 69 OHSS is rare, yet it is a dangerous consequence of gonadotrophin therapy. This syndrome can be reduced but not eliminated with a modification of ovarian stimulation protocols (Forman, 1999; Egbase, 2000). Another potential risk is ovarian cancer. It may increase in frequency later in life due to an increase in ovulation rates after the use of ovarian stimulation. So far, the studies performed have many limitations and it is necessary to perform large multicentre and cooperative studies. A true causal relationship between use of ovulation inducers and increased risk of ovarian cancer has not yet been demonstrated. Conclusions Ovulation stimulation is one of the main pillars of infertility treatment. Gonadotrophins have been used since the 1930s. In the beginning, they were extracted from the pituitary glands of pigs or the serum of mares. These animal extracts were used until the 1950s, when gonadotrophins from the pituitary, extracted from cadavers, or from menopausal urine began to be used. Given the devastating side-effects (Creutzfeld Jacob disease) and the difficulty of extracting pituitary gonadotrophins, urine-derived gonadotrophins were widely used and improved with time. By the 1980s, new formulae were developed, particularly with FSH activity, and by the 1990s, the first pure biological extract of FSH was obtained. The exponential increase in the demand for new assisted reproduction techniques led to the use of recombinant DNA technologies to create recombinant FSH. rfsh is a product that is >99% pure and highly consistent between batches, with no LH activity, and a high specific activity. Gonadotrophin protocols have been tested for monofollicular ovulation as well as for multifollicular stimulations. For monofollicular induction, protocols are adjusted to the needs and characteristics of patients, and the most common are the low-dose step-up or the low-dose step-down protocols. In multifollicular stimulations a combined therapy of pituitary suppression with gonadotrophins is used, widely available for ovulation induction in assisted reproduction. The administration of these medications can be continuous, divided in intervals, or prolonged. In gonadotrophin protocols for either monofollicular of multifollicular stimulations, once the ovarian stimulation has begun, follicular growth can be monitored with regular ultrasounds and/or hormonal testing, mainly for oestradiol in the follicular phase. Recombinant FSH (rfsh) represents a new instrument with which to study the role of gonadotrophins in human folliculogenesis. Initial studies suggest that FSH alone can induce the growth of pre-ovulatory follicles. Many studies have shown that rfsh preparations found in the market can safely and effectively stimulate oocyte development with an improved pregnancy rate (Daya and Gunby, 1999). The experience with rfsh allows its use in research as well as in everyday clinical practice. rfsh seems to be safe and effective for follicular development in anovulatory group II women (WHO, 1973), whereas in anovulatory group I (WHO, 1973), coadministration of LH is mandatory to obtain adequate follicular steroidogenesis. A significant advance in assisted reproduction concerns the adoption of ovulation induction strategies with GnRH analogues, to control the endogenous secretion of LH. These strategies are based on the desensitization of the pituitary, producing hypogonadotrophic hypogonadism for a period of time between 8 and 21 days or longer in some cases. Combined therapy of pituitary suppression with ovarian stimulation using gonadotrophins was used first on women defined as poor responders, but the application of this therapy was extended to assisted reproduction cycles. One of the reasons for the significant improvement in pregnancy rates using analogues is the fact that it avoids cancellations due to an early LH surge, and premature ovulation before obtaining the oocytes. The disadvantages regarding the additional requirement of medication, the increase in gonadotrophin doses, and the cost of the cycle, seem to be positively compensated by an increase in the ability to control the cycle, an increase in the number of oocytes and embryos, and an increase in pregnancy rates for most women. Several protocols to stimulate ovulation with GnRH analogues have been designed, used and compared (Figure 13). Depending on the moment when pituitary suppression starts, there is a short protocol where the administration of the agonist begins at the onset of the menstrual cycle; the ultra-short administration begins later and for a shorter time; the long protocol in which the agonist is administered in the preceding luteal phase; and the ultra-long which is the same as the long protocol but the suppression period is longer. The use of long protocols not only increases pregnancies and live births, it also allows easier programming and simplifies IVF treatments. Regarding the selection of the GnRH analogue, the most generalized option due to its results, is the long protocol with daily subcutaneous injections. Poor responders to classical ovarian stimulation protocols have a lower pregnancy rate. It is important to study the factors that cause the poor response in these patients. There are some tests that can be performed to analyse the response of patients. These tests are based on hormonal content to evaluate the functional ovarian reserve of the patients. Even though the oocyte and Figure 13. GnRH agonists (agnrh) and gonadotrophins: short and ultra-short protocols, long and ultra-long protocols.