Likewise LH and hCG , the alpha subunit of FSH has 92 amino acids (AA). The beta subunit is composed of 111 amino acids with four N-linked glycosylation sites, two on the alpha subunit, added to Asn52 and Asn78, and two on the beta subunit (Asn7 and Asn24) [53, 63]. Thereby, each subunit is attached to two carbohydrate moieties with variable compositions that, in turn, create different isoforms (Fig. 28.5; [3, 53]). These multiple isoforms of FSH differ in their plasma half-lives (ranging from 3 to 4 h) and bioactivity [3] .

Although both sialic acid and sulfonated GalNAc residues modulate the half-lives of human gonadotropins, sialic acid residues are much more common in FSH than sulfonated residues [60]. Increased sialylation enhances FSH metabolic stability by decreasing both glomerular filtration and clearance by sialoglycoprotein receptors in the liver, which is the major site for gonadotropin clearance [64, 65]. It means that the greater the sialic acid content, the longer the hormone remains in circulation [54–56, 60, 64, 65].

Production of different isoforms is controlled by a combination of steroidal feedback and GnRH [55, 66]. The higher the estradiol levels, the lower the FSH sialylation (Table 28.1; [3, 67]). Therefore, the pattern of circulating FSH during the menstrual cycle is dynamic with respect not only to its quantity (concentration) but also to isoform distribution (quality) [58]. The isoform profile is more acidic during early follicular to midfollicular phase, and become more basic shortly before ovulation [3, 58, 68]. These dynamic changes in sialylation are not mimicked by exogenous gonadotropin formulations, and it is unknown whether the absence of such fluctuations during controlled ovarian stimulation (COS) would affect oocyte quality [3] .

Although the LH alpha subunit is identical to that of FSH, the beta subunit contains more amino acids (121 AA) than FSH, a difference that confers its specific biologic activity and is responsible for its interaction with the LH receptor [3] .

Elimination of LH from circulation is modulated by the number of both SO3-GalNAc and sialic acid residues attached to the carbohydrate moieties [64]. LH β-subunits contain a single site of N-linked glycosylation (Asn 30) and less sialic acid residues (only 1 or 2); as such, LH has a short initial half-life of only 20–30 min (Fig. 28.6; [64]). Furthermore, LH molecules with increased number of SO3-4GalNAc disappear faster from the circulation due to binding of sulfonic groups to specific SO3-4GalNAc receptors at the hepatic endothelial cells [54, 59]. In rats, it has been demonstrated that the aforesaid hepatic receptors bind bovine LH with highest affinity only when two or more sulfonated GalNAc residues are present on multiple oligosaccharides. Because this particular carbohydrate structure is also found on human LH, it seems possible that a similar system operates in humans [60].

Likewise FSH, LH shows fluctuations in isoform profile during the menstrual cycle. More basic LH isoforms are seen at midcycle due to considerably decreased sulfonation concomitant with slightly increased sialylation. Both changes increase LH half-life in the circulation, thus explaining the increased levels of serum LH at this period. This change in isoform profile seems to be physiologically important for ovulation triggering [60] .

As already mentioned, the alpha subunit of hCG is identical to those of LH and FSH. Although hCG amino acid sequence is similar to that of LH, a notable difference is the presence of a long carboxyterminal segment with 24 AA containing four sites of O-linked oligosaccharides (Fig. 28.7; [3, 53]). In addition, hCG beta subunits contain two sites of N-linked glycosylation compared with a single site in LH. Due to the higher number of both glycosylation sites and sialic acid residues (approximately 20) than LH, native hCG exhibit a markedly longer terminal half-life in comparison with LH (Table 28.2; [64]) .

The development of gonadotropin preparations began in 1910, when experimental evidence suggested that the pituitary had a role in the regulation of gonadal stems. Crowe et al. were the first to show that partial pituitary ablation resulted in gonadal atrophy in adult dogs and persistence of infantilism in puppies. Two years later, Aschner confirmed these findings and postulated that pituitary function depended upon the function of higher centers in the brain. This author was the first to suggest that the gonads were affected by pituitary extracts and that their use might have clinical applications [2].

In the late 1920s, numerous studies with different species showed that implantation of anterior pituitary tissue from sexually mature females into sexually immature counterparts induced precocious sexual maturity, marked enlargement of the ovaries and superovulation. In contrast, reproductive function was lost after complete pituitary ablation in both sexes [2, 3].

In 1929, Zondek proposed that two hormones were secreted by the pituitary and had stimulatory effects to the gonads. These hormones were named “Prolan A” and “Prolan B” which are now known as FSH and LH, respectively. These authors described the relationship between the pituitary and gonads and the cyclical secretory dynamics of the two gonadotropins in women [2, 3].

Zondek, in collaboration with Ascheim (1927), demonstrated that the blood and urine of pregnant women contained a gonad-stimulating substance capable of inducing both follicular maturation and ovarian stromal luteinization when injected into immature mice. They believed that this substance was produced by the anterior pituitary. Subsequently, it has been shown that this substance was human chorionic gonadotropin (hCG) being produced in the placental tissue of the syncytiotrophoblast [2, 3, 69]. In vitro production of hCG was then possible by culturing placental tissue. hCG was commercially launched in 1931 under the label of Pregnon®, later changed to Pregnyl® [2, 3].

In 1930, following the discovery of hCG, a substance found in the maternal–fetal interface of pregnant mares and extracted from their blood was purified, stabilized as a powder, and sterilized for use in laboratory and clinical studies. This substance was named “pregnant mare’s serum gonadotropin” (PMSG; [3]). Early observations revealed that hCG administered alone in the follicular phase failed to promote follicular development and ovulation , indicating that hCG had no effect in the absence of FSH [2, 70]. In contrast, clinical trials demonstrated that PMSG was able to induce an ovarian response, but attempts to fully induce ovulation produced inconsistent results [71]. Concomitantly, researchers sought other gonadotropic animal extracts to be used in the treatment of infertile patients suffering from gonadotropin insufficiency. In the 1930s, gonadotropins extracted from swine and sheep pituitaries were tested clinically to treat such patients [2].

The concept of using PMSG, hog or sheep pituitary gonadotropins to stimulate follicular development and hCG to trigger ovulation (two-step protocol) was introduced in 1941 [72]. Pituitary animal extracts and PMSG were used in both Europe and the USA until the early 1960s, despite the findings that such treatments resulted in the production of neutralizing antibodies (antihormones), which rendered the ovaries unresponsive to repeated stimulation [2, 3, 73]. Due to the aforementioned effect, PMSG was withdrawn from the market in the early 1970s. Nevertheless, animal-derived gonadotropins were still available in some eastern European countries until 1998 [2].

The recognition that animal gonadotropins induced the production of antihormone antibodies, which could neutralize not only the preparation administered but also endogenous gonadotropins, had been the driven forces of scientific and technological efforts to extract and purify gonadotropins from human sources. In this sense, a special interest group, named “G club,” was formed in 1953 to coordinate and promote the development of specific assay procedures as well as bioassay standards and purification methods to obtain gonadotropin preparations suitable for therapeutic purposes [2].

Human pituitary gonadotropin (hPG) was first isolated in 1958 by Carl Gemzell. Between 1958 and 1988, hPG preparations were successfully used for ovulation induction worldwide [74, 75]. However, it soon became clear that the supply of human pituitaries was too limited to fulfill its constantly growing demand [1–3]. Pituitary glands from ten individuals were needed to yield sufficient quantities of gonadotropin to stimulate one patient for one cycle. In addition, there were constant problems with purification and dose standardization [1]. By the mid—1980s, cases of dementia and death due to iatrogenic Creutzfeldt-Jacob disease (CJD) were identified in Australia, France, and the UK, and were linked to the use of hPG and human pituitary growth hormone. As a consequence, hPG was banned from the market approximately 20 years after its introduction [2, 3].

hMG, or menotropin, was first extracted from the urine of postmenopausal women in 1949 [2]. Urine was originally obtained from an Italian nunnery, and early preparations contained varying amounts of FSH, LH, and hCG in only 5 % pure forms [3]. Improvements in the purification techniques standardized FSH and LH activities to 75 IU for each type of gonadotropin in 1963, as measured by standard in vivo bioassays (Steelman–Pohley assay). The first hMG preparation was registered in Italy in 1950, but clinical trials only started 10 years later [3]. hMG preparations have both FSH and LH activity, but the latter is primarily derived from the hCG component present in postmenopausal urine and concentrated during purification [2, 13, 14]. Sometimes hCG is added to achieve the desired amount of LH-like biological activity [2]. In 1999, purified hMG gonadotropins were introduced, allowing its subcutaneous (SC) administration [3, 4]. At present, both conventional hMG and highly purified hMG (HP-hMG) are commercially available in a FSH:LH ratio of 1:1 [4].

In the 1980s, pure urinary FSH preparations were produced by removing LH with polyclonal antibodies. The production process was essentially passive since LH was separated from the bulk material, and FSH, together with some other urinary proteins, was collected and lyophilized. Despite being a biologically purer urinary gonadotropin, urofollitropin, or purified urinary follicle-stimulating hormone (hFSH-P) still contained high amounts of urinary proteins [76]. Further technological advances made it possible to use highly specific monoclonal antibodies to extract FSH and produce HP-hFSH. The latter became commercially available in 1993 and is available to date. Such preparations contain < 0.1 IU of LH and < 5 % of unidentified urinary proteins. FSH specific activity is approximately 10,000 IU/mg protein compared to 100–150 IU/mg protein in the earlier urinary hMG preparations (Table 28.3). Likewise HP-hMG, the enhanced purity of HP-hFSH enabled SC delivery [3]. Subcutaneous gonadotropin administration represented an important gain for patients. Consistently better tolerability (lower pain at injection site) was reported with SC injections compared with the intramuscular route. More importantly, it allowed self-administration which is more convenient and less time-consuming, as patients need fewer visits to the clinic or hospital for injections [77, 78].

Recombinant technology has met the need for a more reliable source of FSH. Under appropriate conditions, the genes coding for the human FSH alpha subunit and betasubunit have been incorporated into the nuclear DNA of a host cell via a plasmid vector, using spliced DNA strings containing the FSH gene and segments of bacterial DNA [2, 3, 79]. Early recombinant technology focused on producing biological molecules in bacterial cells, usually Escherichia coli, as for insulin production. However, due to the complex structure of human gonadotropins and the need of post-translational glycosylation, which define the degradation time and bioactivity, production of functional FSH was not possible using prokaryotes. Certain mammalian cell lines have been otherwise used to produce recombinant complex proteins, including erythropoietin and gonadotropins. The Chinese hamster ovary (CHO) cell line has been chosen to produce gonadotropins because it is genetically stable, fully characterized and easily transfected with foreign DNA. Furthermore, it can be grown in cell cultures on a large scale and produce adequate levels of biologically active rec-hFSH [2, 79].

In 1995, the first rec-hFSH (follitropin alfa) was licensed for clinical use in the European Union. One year later, a similar rec-hFSH (follitropin beta) was made available [2]. In the manufacturing process of follitropin alfa, two separate vectors, one for each subunit, are used to build the master cell bank of FSH-producing cell line, unlike follitropin beta in which a single vector contains the coding sequences of both subunit genes [79, 80]. The subsequent production steps are similar for both preparations (Fig. 28.8). First, a working cell bank is established by growing cells from a single vial that contains identical cell preparations. An aliquot from the selected clone of CHO cells is grown in T-flasks, then subcultured into roller bottles and allowed to expand for up to 36 days. The cells are then mixed with a suspension of microcarrier beads and transferred to a bioreactor vessel with continuous culture media infusion for an average of 34 days. The cell culture supernatant medium, containing the proteins secreted by the cells, is collected from the bioreactor. The harvested “crude FSH” is stored at 48 °C until purification [2]. Lastly, the protein is purified by chromatography, followed by ultrafiltration. The downstream purification process differs for the two commercially available recombinant FSH preparations. The follitropin beta process uses a series of anion and cation exchange chromatography steps, hydrophobic chromatography and size exclusion chromatography. A similar series of chromatography steps are used in the production of follitropin alfa, in addition to an immunoaffinity step with a specific monoclonal antibody that is similar to the one used in the production of HP-hFSH [2, 79]. Each purification step is rigorously controlled in order to ensure batch-to-batch consistency of the final purified product [2]. While the production of urine-derived gonadotropins is often performed in open, nonsterile environments, the production of rec-hFSH takes place in closed, sterile environments, such as the bioreactor. Both the production and the purification of rec-hFSH are subject to continuous quality control assessments, ensuring a pure, consistent and high-quality product [79]. These same concepts highlighted above are now used in the manufacturing process of other recombinant gonadotropins including LH and hCG [2, 3].

Both recombinant FSH preparations are structurally similar to native FSH. Despite being named follitropin alfa and follitropin beta, each one comprises alpha and beta glycoprotein chains [3]. However, due to the slight differences in their production and purification procedures the preparations are not identical, with variations in posttranslational glycosylation that result in different sialic acid residue compositions and different isoelectric coefficients [3, 81, 82]. Follitropins alfa and beta are similar to the native FSH isoforms found in the blood around mid-cycle (more basic isoforms), but they differ slightly in the charge heterogeneity as follitropin alfa has slightly more acidic glycoforms than follitropin beta [58, 81]. Despite these differences, both preparations have equivalent immunopotency, in vitro biopotency and internal carbohydrate complexity. Immunoassay showed that initial and terminal half-lives after administration of 150 IU recombinant FSH were 2 and 17 h, respectively. Given their intrinsically similar structures, clinical efficacy is expected to be equally similar [3, 81, 83].

Due to the relatively short half-life of FSH (about 1 day), daily FSH injections are needed during the stimulation period to prevent the drop of serum FSH levels below the threshold which cause follicular growth arrest [84]. After each injection, peak serum FSH levels are reached within 10–12 h, and then FSH levels decline until the next injection. Steady state levels are reached only after 3–5 days of treatment, thus dose adjustments before day 5 of stimulation are not advised [85].

A number of technological approaches have been used to develop longer-acting FSH molecules, most of which have involved altering the structure of the FSH molecule itself [85]. Recently, a novel long-acting gonadotropin molecule has been developed by combining rec-hFSH with the C-terminal peptide of hCG using site-directed mutagenesis and gene transfer techniques. It is based on the principle that the highest half-life of hCG is given by the long carboxy terminal segment called C-terminal peptide (CTP) of the beta subunit. The hCG-CTP includes four additional O-linked carbohydrate side chains, each with two terminal sialic acid residues that confer long half-life to hCG [64, 86, 87]. The new molecule has been created using a chimeric gene containing the sequence encoding the CTP fused to the translated sequence of the human FSH beta subunit. The chimera was then transfected with the common glycoprotein alpha subunit and expressed in CHO cells. It has been demonstrated that the presence of the CTP sequence had no significant effect on the assembly or secretion of the intact dimer by stable cell lines. The chimeric recombinant molecule has shown to have similar in vitro receptor binding and steroidogenic activity compared with wild-type FSH, but with significantly enhanced in vivo activity and plasma half-life [85, 88].

The generation of a new CHO cell line expressing the aforementioned FSH hybrid molecule has led to the development of corifollitropin alfa, which was launched in the market in 2010. Corifollitropin alfa is devoid of LH activity. As such, it interacts exclusively with FSH receptors and has a plasma half-life of 65 h [9, 88]. The optimal corifollitropin dose has been calculated to be 100 mcg for women with a body weight ≤ 60 kg and 150 mcg for women with a body weight > 60 kg. From phase II and III studies, it was concluded that a single injection of corifollitropin alfa would replace the first seven daily injections of standard gonadotropins, and that stimulation could be continued with daily FSH injections until the final oocyte maturation had been reached [89]. As the aim of corifollitropin alfa is to simplify treatment and reduce burden associated with multiple injections, it has been developed to be used in GnRH antagonist cycles [85].

Currently, there are three groups of commercially available gonadotropin preparations containing LH activity, that is, (i) urinary hMG, in which LH activity is dependent on hCG rather than on pure LH glycoprotein, (ii) pure LH glycoprotein produced by recombinant technology (lutropin alfa), and (iii) a combination of pure FSH and LH glycoproteins in a fixed ratio of 2:1 also manufactured by recombinant technology (Table 28.4; [3]).

While hMG has been used for ovarian stimulation since 1960, rec-hLH (lutropin alfa) was introduced in the market in the year 2000 for use in women with gonadotropin insufficiency. The manufacturing process of rec-hLH is similar to rec-hFSH. Lutropin alfa is highly pure and has high-biological activity (9000 IU/mg protein; [3]). It is intended to be used subcutaneously in daily injections. Up to date, lutropin alfa is the only recombinant form of human LH developed for use in ovarian stimulation. It is presented in vials of 82.5 IU lyophilized pure glycoprotein powder to be reconstituted with diluent before administration using a conventional syringe and needle (75 IU of lutropin alfa is delivered per vial) [90].

At present, rec-hLH is used not only to support follicular development during COS in hypogonadotropic hypogonadic woman but also in other categories of female infertility [2, 91, 92]. Recombinant LH has three major differences compared to urinary products. First, it has higher purity and specific activity because it is manufactured using recombinant technology. Second, it is associated with better dose precision due to FbM technology that virtually eliminates batch-to-batch variation and will be discussed later in this chapter [2, 6, 93]. Third, LH activity is derived directly from pure LH glycoprotein unlike hMG, in which hCG is concentrated during purification or added to achieve the desired amount of LH-like biological activity [2]. LH and hCG differ in the composition of their carbohydrate moieties which, in turn, affect bioactivity and half-life. As mentioned earlier, LH activity in serum is 30 times higher when hCG is used due to its higher binding affinity to LH receptors. After administration, recombinant human LH is eliminated with a terminal half-life of 9–12 h in contrast to 23–31 h of hCG [64, 94]. It has been shown that the expression of LH/hCG receptor gene, as well as genes involved in the biosynthesis of cholesterol and steroids in granulosa cells, are lower in patients treated with hMG preparations [95]. Such effects are caused by a constant ligand exposure during the follicular phase due to longer half-life and higher binding affinity of hCG compared with rec-hLH. In animal models, down-regulation of LH receptors is maintained for up to 48 h after hMG administration [96]. These findings indicate that the GCs have lower LH-induced cholesterol uptake, a decrease in the novo cholesterol synthesis and a decrease in steroid synthesis, thus explaining the observed lower serum progesterone levels achieved in patients treated with hMG [95, 97]. The clinical implications of these findings, however, have not been fully elucidated [98].

In 2007, a new fixed combination of rec-hFSH and rec-hLH at 2:1 ratio was launched (follitropin alfa + lutropin alfa) as an alternative for those women who need LH supplementation [8]. The 2:1 ratio of FSH and LH in a fixed dose combination was obtained by recombinant technology and vial filling using protein mass (FbM). The use of FbM as opposed of filled-by-bioassay was possible because the specific activity, isoform distribution and sialylation profile of both gonadotropins are highly consistent among manufactured batches [6]. The bioequivalence of rec-hFSH and rec-hLH administrated alone or in combination has been similar [8, 99].

During controlled ovarian stimulation , hCG administration has been the gold standard for ovulation induction as a surrogate for the mid-cycle LH surge for several decades [100] . Due to structural and biological similarities, hCG and LH bind to and activate the same LH/hCG receptor [101]. However, the half-life of hCG is much longer than LH, and serum LH activity of hCG is 30 times higher than LH. Therefore, the luteotropic activity of hCG exerts is markedly higher than LH [94, 102]. After hCG administration, approximately 36 h is required for completion of the meiotic process. In the absence of oocyte retrieval, ovulation will ensue approximately 4 h later [103] .

The first chorionic gonadotropin preparations were developed in 1931 from urine of pregnant women. At present, preparations of urinary hCG are marketed in lyophilized vials of 5000 or 10,000 IU to be used intramuscularly. Although improvements have been achieved in the purification process of urinary hCG, with highly purified presentations being introduced in 1976, urinary hCG is nowadays not recommended for subcutaneous administration. In 2001, hCG preparations using recombinant technology were launched (choriogonadotropin alfa). Recombinant hCG is available in prefilled syringes containing 250 mcg of pure hCG, which is equivalent to approximately 6750 IU of urinary hCG [3]. Recently, a new prefilled pen device has been introduced for administration of rec-hCG. Besides the benefits of higher purity, rec-hCG enables subcutaneous administration, unlike urinary hCG which requires intramuscular administration. Recombinant hCG is thus better tolerated and allows patient self-administration [104]. Nevertheless, the clinical efficacy of both urinary and recombinant preparations to induce final follicular maturation and resumption of oocyte meiosis does not seem to differ [105]. In a Cochrane meta-analysis including 11 randomized controlled trials (RCT) with a total of 1187 women, Youssef et al. compared rec-hCG versus urinary hCG for final oocyte maturation triggering in GnRH agonist down-regulated cycles for in vitro fertilization/intracytoplasmatic sperm injection (IVF/ICSI). There was no evidence of a statistically significant difference between rec-hCG and urinary regarding ongoing pregnancy/live birth rate (6 RCTs: odds ratio [OR] = 1.04, 95 % confidence interval [CI]: 0.79 to 1.37; I²= 0 %), incidence of ovarian hyperstimulation syndrome (OHSS; 3 RCTs: OR = 1.5, 95 % CI: 0.37 to 4.1; I²= 0 %) and number of retrieved oocytes (9 RCTs: Mean difference = − 0.04, 95 % CI: − 0.69 to 0.62; I² = 18 %; [105]) .

A list of gonadotropin preparations currently available for clinical use is provided in Table 28.5 .