Abstract
Ovulatory dysfunction, which is common among women of reproductive age, often results in infertility. Over the last three decades, follicle-stimulating hormone (FSH), in the form of either urinary human menopausal gonadotropin or highly purified urinary FSH (uFSH), has been the mainstay in the treatment of women with ovulatory dysfunction. However, these preparations have several disadvantages, including variable composition, contamination with urinary proteins, and the limited availability of human menopausal urine from which uFSH is extracted. Recombinant human FSH (rhFSH) demonstrates higher purity and specific activity, and is suitable for subcutaneous administration. Additionally, rhFSH has facilitated the development of additional FSH products such as FSH-carboxy terminal peptide that possess different pharmacokinetic and pharmacodynamic properties and may provide more options in the treatment of ovulatory dysfunction and infertility. This article reviews the mechanism of action of FSH in folliculogenesis and ovulation, the current use of FSH in women for the medical management of infertility, and the published clinical experience to date with different rhFSH preparations.
Keywords
Infertility affects more than 6 million couples in the USA, or approximately 10 to 15% of couples in the reproductive population, with 30–40% of female infertility due to anovulatory disorders [1]. The current medical management of infertility frequently involves the use of gonadotropins in women.
Gonadotropin hormones play a central role in the regulation of follicular development and ovulation. Inadequate levels of the gonadotropin, follicle-stimulating hormone (FSH), can result in the absence of ovulation.
Urinary-derived preparations of FSH (uFSH) are extracted from the urine of menopausal women. They have been used successfully to induce ovulation in anovulatory women (World Health Organization [WHO] groups I and II) and to stimulate multifollicular development during ovarian stimulation regimens for in vitro fertilization (IVF) [2]. Although effective, uFSH has several disadvantages, including variable composition, contamination with urinary proteins, and limited supply of raw materials for purification [2,3]. Advances in purification techniques have resulted in the production of highly purified uFSH, which contains over 95% FSH. The introduction of recombinant human FSH (rhFSH) in the early 1990s has overcome some of the limitations of urinary-derived preparations. Recombinant human FSH demonstrates high potency, uniform composition, and high specific activity [4–7].
This article reviews the mechanism of action of FSH in folliculogenesis and ovulation, the current use of FSH in women for the medical management of infertility, and published clinical experience to date with the use of different FSH preparations in women. Recent findings of studies evaluating the use of a pen device for self-administration of rhFSH and novel long-lasting rhFSH agonists are also discussed.
Role of FSH in folliculogenesis
Human FSH is composed of a family of complex heterodimeric proteins consisting of two subunits, designated the α and the β subunit. The various proteins in the human FSH family differ in their degree of glycosylation and sialic acid content [8]. Although the role of FSH in the early stages of folliculogenesis has not been fully elucidated, FSH is required for the later stages of follicular development. Apart from stimulating the growth of the preantral follicle, FSH also stimulates maturation of the preantral follicle into the preovulatory follicle by initiating granulosa cell aromatase activity and enzymes involved in progesterone biosynthesis. FSH also induces receptors for luteinizing hormone (LH) on granulosa cells, thereby allowing ovulation and development of the corpus luteum in response to the mid-cycle LH surge [9].
FSH stimulation plays a critical role in follicular recruitment and dominant follicle selection [10]. During the normal ovulatory cycle, increasing FSH levels in the luteo-follicular transition phase stimulates the continued growth of approximately 5 to 10 antral follicles per ovary [11]. Each of these growing follicles has the potential for continued growth if the threshold requirement for FSH stimulation is achieved. As FSH levels decrease in response to negative feedback from estradiol and inhibin levels in the late follicular stage, only one dominant follicle with increased sensitivity to FSH continues to grow, while the remaining follicles enter into atresia. The administration of exogenous FSH widens the ‘FSH window,’ allowing the pool of maturing recruited follicles to be rescued from atresia and attain dominance [12].
Numerous preparations of FSH have been developed over the years that differ in composition, purity, safety, and efficacy (Table 1). Initial FSH preparations, referred to as human menopausal gonadotropin (hMG), were derived from the urine of postmenopausal women. Although hMG preparations are effective, they contain significant amounts of LH and other urinary proteins, which may contribute to complications such as injection-site pain and systemic allergic reaction [13,14]. Advances in the purification of FSH enabled the production of other urinary-derived FSH preparations (urofollitropin and highly purified urofollitropin). These preparations possess higher specific activity with significantly reduced or negligible quantities of LH (<5%, Metrodin-HP® [Serono International, S.A, Geneva, Switzerland]; <2%, Bravelle® [Ferring Pharmaceuticals]) and yield higher pregnancy rates than hMG, particularly when used for ovarian stimulation in IVF [15–17]. However, batch-to-batch variability in FSH isoform content and limited quantities of material for purification remain considerable disadvantages for urinary preparations.
Currently available FSH products.
FSH: Follicle-stimulating hormone; LH: Luteinizing hormone; rhFSH: Recombinant human follicle-stimulating hormone; uFSH: Urinary follicle-stimulating hormone; uLH: Urinary luteinizing hormone.
Adapted from Huirne et al. (2004) [2] by kind permission of Adis International.
Many of the clinical limitations of uFSH were overcome by the development of rhFSH. Recombinant techniques used in the preparation of rhFSH have resulted in high purity, high specific activity (10,000 international units [IU] FSH/mg), minimal variation in composition, and improved efficacy [5,6,18]. Two rhFSH preparations are currently available in the form of follitropin α (Gonal-F®, Serono International S.A., Switzerland) and follitropin β (Puregon®/Follistim®, Organon, The Netherlands). Although the amino acid sequence of rhFSH is identical to that of human pituitary FSH, the two recombinant products differ from each other and from human pituitary FSH in their degree of glycosylation and sialylation. Highly sialylated isoforms are more acidic, resulting in longer half-life, lower receptor binding in in vitro receptor binding assays, and lower in vitro bioactivity than less sialylated/basic isoforms (pH 5.5–6.0) [8,18]. The biologic activity of rhFSH and uFSH, as assessed by determining rat ovarian weight gain in response to FSH administration, are essentially equivalent [18].
Use of FSH for the medical management of female infertility
Anovulatory infertility
The goal of ovarian stimulation strategies in anovulatory women is to stimulate the growth and ovulation of a single dominant follicle. A variety of treatment protocols that involve stepwise dosing regimens for ovarian stimulation have been developed. The original (standard) ‘step-up’ protocol consists of administering a starting dose of FSH 150 IU/day. This protocol is often associated with multifollicular development, resulting in a high rate of complications such as ovarian hyperstimulation syndrome (OHSS), and multiple gestations. Subsequently, a low-dose ‘step-up’ protocol was developed that involves the administration of the initial subcutaneous or intramuscular dose of FSH 50–75 IU/day. If no response is observed on ultrasonography after 14 days, the dose of FSH is increased at weekly intervals, using 25 or 37.5 IU increments. This protocol has a similar pregnancy rate as the standard step-up protocol but a lower incidence of multifollicular development and OHSS. Although effective at stimulating follicular development, both step-up protocols deviate from physiologic observations. Due to the negative feedback of estradiol and inhibin, the levels of FSH decrease after the recruitment of a cohort of follicles. This may be partly responsible, in addition to intraovarian regulators, for the selection of the dominant follicle.
In contrast, the ‘step-down’ protocol consists of FSH 150 IU/day administered shortly after spontaneous or progesterone-induced menses and maintained until a dominant follicle of ≥10 mm is detected by transvaginal ultrasonography. If ovarian response is not observed after 3–5 days, the dose may be increased until follicular development occurs. The dose is then decreased in a stepwise manner to 112.5 IU/day for 3 days and then to 75 IU/day. The latter dose is maintained until human chorionic gonadotropin (hCG) 5000 to 10,000 IU is administered to induce ovulation.
The safety and efficacy of step-up and step-down protocols using rhFSH (follitropin β) were recently compared in anovulatory women (n = 83) [19]. In this study, the low-dose step-up protocol was more efficient in obtaining monofollicular development and ovulation than the step-down protocol (68.2% of the 85 cycles in the step-up group vs 32% of the 72 cycles in the step-down group, p < 0.0001) [19]. The duration of ovarian stimulation was longer in the step-up group (15.2 ± 7.0 days in step-up vs 9.7 ± 3.1 days in step-down, p < 0.001). Although ovulation occurred more frequently in patients receiving the step-up procedure as compared with the step-down procedure (70.3% of the cycles in the step-up vs 51.3% in the step-down procedure, p < 0.01), the cumulative rate of clinical gestations during the study did not differ between the two groups (38.6% in the step-up vs 30.8% in the step-down procedure) [19].
rhFSH has demonstrated efficacy in stimulating follicular growth in patients with clomiphene citrate-resistant polycystic ovary syndrome (PCOS) [20–24]. In a pilot study of 11 women with clomiphene citrate-resistant PCOS, Hayden and colleagues found that low-dose rhFSH (follitropin β) was effective in inducing follicular response [24]. These findings were confirmed in a larger study comparing the efficacy of rhFSH (follitropin β) and uFSH (Metrodin) using a low-dose step-up regimen in 172 women with clomiphene citrate-resistant normogonadotropic chronic anovulation [23]. Although rhFSH was more efficient, there was no difference in the cumulative ovulation rates after three treatment cycles between the treatment groups. In a review of four randomized trials comparing rhFSH with uFSH in women with clomiphene citrate-resistant PCOS, Bayram and colleagues reported no significant differences in ovulation, pregnancy, miscarriage, or multiple gestation rates between rhFSH and uFSH [25].
More recently, Szilagyi and colleagues compared the efficacy and safety of rhFSH (follitropin α) and uFSH (Metrodin) in a low-dose step-up protocol for ovulation induction in 20 clomiphene citrate-resistant infertile PCOS patients [21]. In this study, rhFSH resulted in higher pregnancy rates than uFSH. All six pregnancies induced in this study were in the rhFSH group; two ended with miscarriage.
In another study, Lopez and colleagues compared the efficacy and safety of clomiphene citrate and low-dose rhFSH as first-line pharmacologic therapy for 76 infertile patients with PCOS [20]. This study is the first to compare clomiphene citrate to FSH as first-line treatment in women with PCOS. Patients were randomized to receive clomiphene citrate (50–150 mg/day for 5 days, n = 38) or rhFSH (n = 38) in a chronic, low-dose, step-up protocol (daily starting dose 75 IU). After three consecutive cycles, the cumulative pregnancy rate was higher for rhFSH than with clomiphene citrate (43% rhFSH vs 24% clomiphene citrate, p = 0.06). A multicenter European study is currently underway to confirm these results; results from this trial will likely change the way treatment decisions are made in women with PCOS.
Assisted reproductive therapy
IVF with or without intracytoplasmic sperm injection (ICSI) is the most widely practiced form of assisted reproductive technology (ART). In contrast to ovulation-induction regimens for anovulatory infertility, the primary goal of ART is to obtain a high number of oocytes for IVF and intrauterine transfer, or cryopreservation of embryos. Although a number of regimens for ART have been developed, regimens that combine gonadotropins with gonadotropin-releasing hormone (GnRH) agonists have demonstrated superiority over gonadotropin-alone regimens [2]. Pituitary desensitization induced by the addition of a GnRH agonist results in improved folliculogenesis and inhibits the spontaneous LH surge. In clinical trials, the stimulation regimens that combine GnRH agonist with gonadotropins resulted in fewer cancelled cycles, higher numbers of oocytes, and improved pregnancy rates compared with gonadotropin-alone regimens [2]. The most common form of pituitary desensitization, referred to as the ‘long protocol,’ involves the administration of the GnRH agonist in any one of three different protocols. GnRH agonist may be initiated either from the midluteal phase of the previous cycle; while taking oral contraceptive pills; or from cycle day 1 for 12–14 days until pituitary suppression is achieved. When pituitary suppression is achieved, FSH is administered to begin the stimulation phase. The dose of FSH can be adjusted for the individual patient based upon her ovarian reserve and response.
More recently, several novel strategies including the addition of GnRH antagonists or low-dose hCG to stimulation protocols have been investigated in attempts to further improve the safety and efficacy of ART. In large randomized clinical trials comparing daily GnRH antagonist administration with a standard long-protocol regimen in IVF patients, the GnRH antagonist shortened treatment by 1–2 days; however, fewer oocytes were recovered compared with the long protocol [26,27]. A recent meta-analysis of five large randomized trials comparing GnRH antagonists with GnRH agonists for controlled ovarian hyperstimulation (COH) in ART found a significantly lower rate of pregnancy in those treated with GnRH antagonists (odds ratio [OR]: 0.79; 95% confidence interval [CI] 0.63–0.99) [28].
The effect of adding LH activity in the form of low-dose hCG to stimulation protocols has been examined by several research groups. Filicori and colleagues randomly assigned patients undergoing IVF to standard hMG/rhFSH stimulation or to hMG/rhFSH stimulation with a switch to hCG (200 IU/day) when at least six follicles reached 12 mm in diameter [29]. The length of stimulation and the total IU of gonadotropins were significantly reduced in the hCG group. Switching to hCG resulted in continued growth of the larger follicles and to a reduction in the number of smaller follicles. The number of large follicles, oocytes obtained, number of embryos, and pregnancy rates were comparable. At the time of retrieval, fewer small follicles and significantly lower estradiol levels were observed in the hCG group, suggesting a more physiologic response.
In another study women were randomly assigned to one of three stimulation regimens for IVF: rhFSH with late follicular phase hCG (200 IU/day) plus GnRH antagonist, rhFSH with GnRH antagonist, or the midluteal downregulation GnRH agonist long protocol with rhFSH [30]. A lower dose of gonadotropins was used in the hCG group, but in contrast to the previous study, the peak estradiol level was higher in this group. The number of oocytes and the rates of implantation and pregnancy were comparable among the three study groups. The incidence of OHSS was also similar in the three groups.
In a similarly designed study (prospective randomized, same three protocols), Kyono and colleagues found that significantly fewer ampules of gonadotropins were used in the hCG group [31]. The number of follicles, fertilization rate, and the number and quality of follicles were comparable among the three groups. Pregnancy rate was the highest in the hCG group (61.9 vs 47.1 and 42.1%) [31]. The incidence of OHSS was significantly reduced when late follicular-phase hCG was used to maintain follicle growth.
Clinical experience with FSH in ART
Recent retrospective and prospective comparisons of rhFSH with highly purified uFSH have shown that rhFSH is more effective in stimulating follicular development with no difference in the risk of OHSS [5,6,32–34]. In a large-scale, prospective, randomized study comparing rhFSH and uFSH in the treatment of 981 women, rhFSH resulted in more oocytes retrieved (10.84 vs 8.95, p < 0.0001) and higher ongoing pregnancy rates (25.7 vs 20.4%, p = 0.05) than uFSH [5]. Moreover, total FSH consumption (2138 vs 2385 IU, p < 0.0001) and treatment duration (10.7 vs 11.3 days, p<0.0001) were lower with rhFSH compared with uFSH [5]. These findings were confirmed in a meta-analysis of 18 randomized studies of ovarian stimulation for IVF using uFSH or rhFSH [35].
In contrast, no significant differences in the mean number of oocytes retrieved, ongoing clinical pregnancies, or live births were observed between highly purified uFSH (Bravelle) and rhFSH (follitropin β) in two randomized, parallel group, multicenter studies in infertile women undergoing ovarian stimulation for IVF [36,37]. Similarly, a recent meta-analysis of five trials comparing the effectiveness of hMG with rhFSH in ovarian stimulation protocols for IVF treatment cycles, with or without ICSI, found no evidence of a difference between hMG and rhFSH in ongoing pregnancy or live birth per woman [38]. In addition, no differences were observed in FSH dose, oocytes retrieved, and the rates of miscarriage or multiple pregnancies.
One of the advantages of the high purity of rhFSH is the improved safety and tolerability, compared with uFSH preparations. Safety data from large randomized clinical trials of rhFSH and uFSH in ART [5,6,33,39] and anovulatory infertility [21–24] have shown that rhFSH has improved tolerability compared with uFSH. In these studies, rhFSH has consistently demonstrated reduced immunogenicity and better overall tolerability than uFSH preparations with respect to injection-site reactions and systemic allergic reactions. However, no statistical differences have been noted in the incidence of OHSS between rhFSH and uFSH in these studies. Data from a recent meta-analysis of five studies comparing the use of a long protocol of rhFSH with that of uFSH for ovulatory stimulation in IVF showed no difference in the occurrence of OHSS after hMG use when compared with rhFSH (relative risk [RR] 1.5% hMG vs 1.0% rhFSH, CI 0.56–3.73) [38].
The safety and efficacy of the two different rhFSH preparations have been compared in women undergoing ovarian stimulation and IVF. Brinsden and colleagues compared the safety and efficacy of follitropin α and follitropin β in 44 infertile women undergoing IVF and embryo transfer [40]. In this study, no statistically significant differences were observed in the number of mature follicles or oocytes retrieved. The number of clinical pregnancies was also similar between follitropin α and follitropin β (32 vs 27%, respectively). Similar findings were reported by Harlin and colleagues in a second study comparing the effectiveness of follitropin α and β in 296 IVF cycles [41]. In this study, clinical pregnancy rate was similar, 29.1% for follitropin α and 28.1% for follitropin β. In addition, no difference was observed in endometrial response, estradiol response, number of smaller (12–15 mm) or larger (>15 mm) follicles, and number of oocytes retrieved between the two rhFSH preparations.
Recently, three randomized studies have evaluated the efficacy and convenience of a pen device delivering follitropin β in women undergoing ovarian stimulation. An open-label multicenter study examined the safety, effectiveness, and ease of use of a follitropin β pen device in 60 women undergoing COH for IVF, with or without ICSI [42]. Of the 60 women who participated in the study, 98.3% rated the overall experience of self-injecting with the follitropin-β-containing pen device as ‘very good’ to ‘good’ on day six of treatment.
The ease of use, safety, and efficacy of the follitropin β pen device was also evaluated in 43 clomiphene citrate-resistant anovulatory women undergoing ovulation induction [43]. In this study, 100% of the patients rated the overall experience of self-administering with the follitropin β pen device as ‘very good’ to ‘good.’ The mean duration and total amount of follitropin β were 11.4 ± 4.2 days and 1070.3 ± 580.3 IU, respectively. The ovulation rate was 95% and the biochemical and ongoing pregnancy rates per attempt were 34.9 and 30.2%, respectively. Three of the 43 patients in this study experienced serious adverse events that consisted of asthma, OHSS, and pain [43].
The efficacy and convenience of the follitropin β pen device has also been compared with a conventional syringe delivering follitropin α in 200 women undergoing IVF [44]. In this study, the follitropin β pen device was found to be safe and easy, more convenient, and less painful for the patient. The average duration, total dose of rhFSH, and number of oocyte complexes retrieved were 10.8/12.0 days (p = 0.001), 1880/2226 IU (p<0.001), and 15.2/13.1, respectively, in the pen device and conventional syringe groups [44]. The findings of this study differ from previous studies that reported higher injection-site reactions of pain with follitropin β compared with follitropin α, both of which were administered with a conventional syringe and needle [40].
Prior to the introduction of the follitropin-β-containing pen device 6 years ago, all gonadotropin injections were given with traditional needle and syringe. There is now a second pen device on the market in some countries (USA, Australia, Switzerland, Sweden, and The Netherlands) that comes pre-filled with recombinant follitropin α revised formulation female (RFF). This pen device is filled by mass, a change from the previously conventional method of dispensing based on IU of biologic activity as assessed by determining rat ovarian weight gain in response to gonadotropin administration. The follitropin α RFF pen device is available in three sizes, filled with 30 μg (415 IU), 41 μg (568 IU), or 75 μg (1,026 IU) of follitropin α, intended to deliver at least 300 IU in 0.5 ml, 450 IU in 0.75 ml, or 900 IU in 1.5 ml, respectively. It is not reusable.
Executive summary
Follicle-stimulating hormone (FSH) plays a critical role in follicular recruitment and dominant follicle selection. The administration of exogenous FSH widens the ‘FSH window,’ allowing the pool of maturing recruited follicles to be rescued from atresia and attain dominance.
Numerous preparations of FSH are available that differ in composition, purity, and safety. Some disadvantages have been reported with urinary-derived FSH preparations (uFSH), including variable composition, local adverse events, immunologic reactions, and limited availability of human menopausal urine from which FSH is extracted.
Some of these limitations have been overcome by the introduction of recombinant human FSH (rhFSH), which possesses high purity, high specific activity, and improved batch-to-batch consistency compared with uFSH.
Two similar but different rhFSH preparations are currently available in the forms of follitropin α (Gonal-F®) and follitropin β (Puregon®/Follistim®).
In anovulatory infertility, a variety of treatment protocols, including the original (standard) step-up, the low-dose step-up, and the step-down protocols, have been developed to stimulate follicular growth and ovulation.
In a comparative study of the low-dose step-up and step-down protocols using rhFSH (follitropin β) in women with clomiphene citrate (CC)-resistant polycystic ovarian syndrome (PCOS), the low-dose step-up protocol was more efficient in obtaining a monofollicular development and ovulation than the step-down protocol, but with a similar cumulative rate of clinical gestations.
For in vitro fertilization (IVF), compared with gonadotropin-alone regimens, combination gonadotropin-releasing hormone (GnRH) agonist/gonadotropin regimens have demonstrated superiority, resulting in fewer cancelled cycles, higher numbers of oocytes, and improved pregnancy rates.
The addition of GnRH antagonists or low-dose hCG to stimulation protocols have been investigated in attempts to further improve the safety and efficiency of assisted reproductive technology (ART). In large randomized clinical trials comparing a daily GnRH antagonist regimen and standard long protocol in IVF patients, the GnRH antagonist regimen shortened treatment by 1–2 days and reduced the incidence of ovarian hyperstimulation syndrome (OHSS); however, fewer oocytes were retrieved and lower clinical pregnancy rates were achieved with the GnRH antagonist regimens compared with the GnRH agonist long-protocol regimens.
Numerous randomized trials comparing rhFSH and uFSH preparations have shown that rhFSH is more effective than uFSH for ovulation induction in women with PCOS and those undergoing ovarian stimulation for IVF with respect to oocytes yield, FSH consumption, and days of stimulation.
Data from two meta-analyses confirmed these findings and found a statistically significant increase in clinical pregnancy rates with rhFSH compared with uFSH, when used in ovarian stimulation protocols for IVF.
Recent meta-analyses of trials comparing hMG with rhFSH have reported no evidence of superiority in the clinical pregnancy rate for rhFSH.
Randomized studies evaluating the efficacy and convenience of a pen device delivering follitropin β in women undergoing ovarian stimulation have reported a positive overall experience of self-administration with the follitropin β pen device and was found to be safe and easy, more convenient, and less painful for the patient.
The development of an rhFSH agonist with extended half-life could reduce the number of injections while maintaining biologic effectiveness.
Two long-acting rhFSH agonists have been developed by fusing the carboxyterminal peptide (CTP) of hCG to native rhFSH either as a heterodimer in which the hCG is covalently bound to the β subunit or as a contiguous peptide in which CTP is used as a tether and covalently links the C-terminus of the β chain to the N-terminus of the α chain.
In clinical studies, a single injection of FSH-CTP induced follicular growth and resulted in a successful pregnancy and live birth. Further studies are needed to determine the immunogenicity of long-acting rhFSH analogs and their effects on clinical parameters such as folliculogenesis, oocyte quality, embryo development, implantation and pregnancy, and spermatogenesis.
Conclusion
FSH-containing preparations have been shown to be effective for the induction of follicular development in anovulatory women. Recombinant FSH has some advantages over urinary preparations, including high specificity and suitability for subcutaneous administration. The introduction of rhFSH has also facilitated the development of more patient-friendly delivery systems for rhFSH products that may reduce the anxiety of self-administration and novel rhFSH agonists with different potencies and duration of action. A novel pen device for the administration of follitropin β is safe and easy to use and has demonstrated efficacy in the induction of follicular development in women with PCOS and for COH in IVF. Finally, the development of rhFSH agonists with an extended half-life may eventually allow for a reduction in the number of injections required to provide biologic effectiveness and the development of more simplified approaches for ovarian stimulation.
Future perspective
The introduction of rhFSH has facilitated the development of novel FSH compounds with different potencies and duration of action. The relatively short half-life of rhFSH requires daily injections for 8 to 12 consecutive days to stimulate folliculogenesis in women. The development of an rhFSH with an extended half-life can reduce the number of injections while maintaining effectiveness. Two long-acting gonadotropin agonists have been developed by fusing the carboxyterminal peptide (CTP) of hCG to native rhFSH, either as a heterodimer in which the hCG is covalently bound to the β subunit [45] or as a contiguous peptide in which CTP is used as a tether and covalently links the C-terminus of the beta chain to the N-terminus of the α chain [46]. In healthy female volunteers, administration of a single dose (15, 30, or 60 μg) of FSH–CTP (heterodimer) induced multiple follicular growth accompanied by a dose-dependent rise in serum inhibin-B concentrations [47]. In a subsequent study, pregnancy and live birth were achieved after ovarian stimulation with a single subcutaneous injection of rhFSH–CTP 180 μg [48]. Further studies are needed to determine the immunogenicity of long-acting rhFSH analogues, and their effects on clinical parameters such as folliculogenesis, oocyte quality, embryo development, implantation and pregnancy, and spermatogenesis.