Medical brain notes 37

  Follicle Stimulating Hormone INTRODUCTION   About 15% of all couples experience infertility at some time during their reproductive lives. Increasingly, infertility can be treated by the use of assisted reproductive technologies, such as in vitro fertilization (IVF), gamete intra-fallopian transfer, and intracytoplasmic sperm injection. Gonadotropin treatment to increase the number of oocytes is a common element of these programs. A major cause for female infertility is chronic anovulation. Patients suffering from this condition are also treated with gonadotropins with the aim to achieve monofollicular development.   Gonadotropin preparations for infertility treat-ment are traditionally derived from postmenopausal urine. The urinary preparations contain follicle-sti-mulating hormone (FSH), but are typically less than 5% pure. The preparations also contain luteinizing hormone (LH) as a contaminant. Recombinant DNA technology allows the reproducible manufacturing of FSH pr...

  BIOLOGICAL ROLE   The primary function of the glycoprotein hormone FSH in the female is the regulation of follicle growth. FSH is produced and secreted by the anterior lobe of the pituitary, a gland at the base of the brain. Its target is the FSH receptor at the surface of the granulosa cells that surround the oocyte. FSH acts synergistically with oestrogens and LH to stimulate proliferation of these granulosa cells, which leads to follicular growth. This process explains why deficient endogenous production of FSH may cause infertility.

  CHEMICAL DESCRIPTION   FSH belongs to a family of structurally related glyco-proteins which includes LH, chorionic gonadotropin and thyroid-stimulating hormone. Each hormone is a dimeric protein consisting of two non-covalently associated glycoprotein subunits, denoted  a  and  b . The α-subunit is identical for all these gonadotropins, and it is the β-subunit that provides each hormone with its specific biological function.   The glycoprotein subunits of FSH consist of two polypeptide backbones with carbohydrate side chains attached to the two asparagine (Asn) amino acid residues on each subunit. The oligosaccharides are attached to Asn-52 and Asn-78 on the α-subunit (92 amino acids), and to Asn-7 and Asn-24 on the β-subunit (111 amino acids). The glycoprotein FSH has a molecular mass of approximately 35 kDa. For the FSH preparation to be biologically active, the two subunits must be correctly assembled into their three-dimensional dimeric protein struct...

  PRODUCTION OF RECOMBINANT FSH   The genes coding for the human FSH α-subunit and β-subunit were inserted in cloning vectors (plasmids)to enable efficient transfer into recipient cells. These vectors also contained promoters that could direct transcription of foreign genes in recipient cells. CHO cells were selected as recipient cells since they wereeasily transfected with foreign DNA, and are capable of synthesizing glycoproteins. Furthermore they could be grown in cell cultures on a large scale. To construct a FSH-producing cell line NV Organon, the manu-facturer of Puregon /Follistim , used one single vector containing the coding sequences for both subunit genes (Olijve, 1996). Merck Serono S.A., the manufacturer of Gonal-F , used two separate vectors, one for each subunit gene (Howles, 1996). Following transfection, a genetically stable transformant produ-cing biologically active recombinant FSH was iso-lated. For the CHO cell line used for manufacturing Puregon /Follisti...

  ISOHORMONES   Structural Characteristics   As explained above, FSH exists in many distinct molecular forms (isohormones), with identical poly-peptide backbones but diffences in oligosaccharide structure, in particular in the degree of terminal sialylation. These isohormones can be separated by chromatofocusing or isoelectric focusing on the basis of their different isoelectric points [pI, as has been demonstrated for follotropin  b  (de Leeuw et al., 1996)] (Fig. 2). The typical pattern for FSH indicates an isohormone distribution between pI values of 6 and 4. To obtain structural information at the subunit level, the two subunits were separated by RP-HPLC and treated to release the N-linked carbohydrate side-chains. Fractions with low pI values (acidic fractions) displayed a high content of tri- and tetrasialo oligosaccharides and a low content of neutral and monosialo oligosaccharides. For fractions with a high pI (basic fractions) value the reverse was foun...

  Biological Properties of Recombinant FSH Isohormones   A FSH preparation can be characterized with four essentially different assays, each having its own specific merits: ( i ) The immunoassay determines FSH-specific structural features and provides a relative measure for the quantity of FSH. ( ii ) The receptor binding assay provides information on the proper conformation for interaction with the FSH receptor. Receptor binding studies with calf testis membranes have shown that FSH isoform activity in follitropin  b  decreases when going from high to low pI isoforms. ( iii ) The in vitro bioassay measures the capability of FSH to transduce signals into target cells (the intrinsic bioactivity). The in vitro bioactivity, assessed in the rat Sertoli cell bioassay, also decreases when going from high to low pI isoforms. ( iv ) The in vivo bioassay provides the overall bioactivity of a FSH preparation. It is determined by the number of molecules, the plasma residence ti...

  Pharmacokinetic Behavior of Recombinant FSH Isohormones   The pharmacokinetic behavior of follitropin  b  and its isohormones was investigated in Beagle dogs that were given an intramuscular bolus injection of a number of FSH isohormone fractions, each with a specific pI value. With a decrease in pI value from 5.49 (basic) to 4.27 (acidic), the AUC increased and the clearance decreased, each more than 10-fold (Fig. 3). A more than twofold difference in elimination half-life between the most acidic and the most basic FSH isohormone fraction was calculated. The absorption rate of the two most acidic isoforms was higher than the absorption rates of all other isoforms. The AUC and the clearance for the follitropin  b  preparation, being a mixture of all isohormone fractions, corre-sponded with the centre of the isohormone profile (Fig. 3). In contrast, the elimination of the follitropin  b  preparation occurred at a rate similar to that of the most ...

  PHARMACEUTICAL FORMULATIONS   Recombinant FSH preparations distinguish them-selves by their high purity (at least 97%) for example from urinary FSH preparations, which typically have a purity of less than 5%. Pure proteins are, however, relatively unstable and are generally lyophilized, unless some specific stabilizing measures can be taken. FSH preparations are available in different strengths and presentation forms, both as freeze-dried products (powder, cake, lyosphere) and as solution for injection (Table 1). Lyospheres are frozen drops of aqueous solution, which are freeze-dried in bulk and subsequently put in ampoules. Compared to the traditional freeze-dried cake formulation, lyospheres have the advantage of high dose uni-formity, less adsorption to the glass walls of the ampoule, instantaneous dissolution, and in case of FSH, improved stability. Follitropin  a  was originally formulated with sucrose (bulking agent, lyoprotec-tant), sodium dihydrogen phospha...

  CLINICAL ASPECTS   Both recombinant FSH products on the market have been approved for two female indications. The first indication is anovulation (including polycystic ovar-ian disease) in women who are unresponsive to clomiphene citrate. The second indication is con-trolled ovarian hyperstimulation to induce the devel-opment of multiple follicles in medically assisted reproduction programs, such as IVF and embryo transfer. In addition, recombinant FSH may be used in men with congenital or required hypogonadotropic hypogonadism to stimulate spermatogenesis.   In anovulatory infertility in females, FSH treat-ment aims for the development of a single follicle, whereas IVF FSH treatment is aimed at multifollicular development. For the treatment of anovulatory patients it is recommended to start Puregon. Follistim treatment with 50 IU per day for 7 to 14 days and gradually increase dosing with steps of 50 IU if no sufficient response is seen. This gradual dose-increasing sc...