Inhibins as Diagnostic Markers in Human Reproduction

Anastasia Tsigkou*; Stefano Luisi*; Fernando M. Reis†; Felice Petraglia*,1petraglia@unisi.it    * Chair of Obsterics and Gynecology, Department of Pediatrics, Obstetrics and Reproductive Medicine, University of Siena, Policlinico, S. Maria alle Scotte Viale Bracci, 53100 Siena, Italy
† Department of Obstetrics and Gynecology, University of Minas Gerais, Belo Horizonte, Brazil
1 Corresponding author: Felice Petraglia

1 Abstract

Over the past 75 years, many publications have focused on measurement of inhibin concentration and/or activity in biological samples in order to understand its role in physiology and disease. This chapter highlights the accomplishments within this area of research over the past decade including development of specific inhibin assays. Inhibin A is a marker of dominant follicle and corpus luteum activity and decreases in polycystic ovary syndrome (PCOS). Inhibin A increases in gestational diseases such as pre-eclampsia and fetal Down's syndrome, and this increase in inhibin A improves early diagnosis of both conditions. The measurement of inhibin A in women with threatened abortion provides useful information about the likelihood of pregnancy loss. Inhibin B increases markedly in women with granulosa cell tumor and appears closely related to gametogenesis in men, that is, reflecting Sertoli cell activity. On the contrary, Inhibin B decreases in women with declining ovarian function and correlates with female response to ovulation induction. This review evaluates the biochemical significance of inhibins including their use in clinical practice.

2 History and Background

Inhibin is a gonadal hormone that exerts specific negative feedback on pituitary secretion of follicle stimulating hormone (FSH). The existence of inhibin was postulated in 1923 by Mottram and Cramer who observed hypertrophy of pituitary cells after irradiation of the testis of adult rats [1]. McCullagh [2] subsequently investigated this phenomenon by injecting a water-soluble extract from bovine testes and found that it could inhibit hypertrophy of the pituitary and thus named the active, water-soluble principle “inhibin” [2].

The concept of inhibin was refined after the discovery of two separate pituitary hormones, FSH and luteinizing hormone (LH), which together regulate the gonadal function. Klinefelter et al. [3] observed that serum levels of FSH were elevated in oligo- or azoospermic men. Heller and Nelson [4] suggested that the increased serum levels of FSH were caused by a lack of utilization of FSH by the defective seminiferous tubules. Some groups [5, 6] showed that the injection of water-soluble testicular extracts into rats could suppress peripheral FSH levels. de Jong and Sharpe [7] also found FSH-suppressing activity in ovarian follicular fluid. Little, however, was understood about the mechanism of action of inhibin on pituitary cells until Franchimont et al. [8] demonstrated that the addition of inhibin-containing preparations to pituitary cells caused an increase in the secretion of cGMP and a decrease in cAMP secretion. This finding was then followed by Jenner [9] who speculated that the decrease in cAMP in incubated hemipituitaries is followed by a decrease in the synthesis of FSH.

The combination of an FSH radioimmunoassay (RIA) and an in vitro primary pituitary cell culture led to the purification of inhibin from bovine [10] and porcine [11–13] follicular fluids. Cloning established the protein sequence of inhibin as a 32-kDa dimeric glycosylated molecule consisting of an α and a β subunit. It soon became clear that there were two forms of inhibin. Each contains a common α subunit associated with either βA (inhibin A) or βB (inhibin B) subunits that also possess a high degree of homology (Table 1) [12]. Inhibin A and inhibin B were found to be equipotent in the rat pituitary cell bioassay [14], but in the sheep pituitary cell bioassay [10], inhibin A was much more potent than inhibin B. During the purification in bovine, it became clear that there was a large amount of free α chain inhibin that had no bioactivity [10] and could potentially interfere with quantitative assay unless the models were specifically configured to ignore its presence. Initially, inhibin was identifiable as a distinct entity only by bioassays [15–18]. These, of course, were of limited specificity, and generally unsuitable for use with complex body fluids. The development of highly specific and sensitive immunoassays proved essential in advancing the study of inhibin in physiologic as well as pathophysiologic conditions.

The original Monash RIA [19] was widely used in physiological studies before it was realized that it was detecting mostly free α subunits rather than dimeric bioactive inhibin forms in human body fluids [20]. The breakthrough in our understanding of the biology of inhibin came when Groome and collaborators established a specific immunoassay for the measurement of dimeric inhibin A in the menstrual cycle [21]. An assay for measurement of dimeric inhibin B was also subsequently developed by this group [22]. Both assays have been found to be useful for many clinical applications, some of which are detailed below.

During the purification of inhibin from porcine follicular fluid, two research groups isolated a protein that displayed FSH-releasing activity from the gonadotrophs in vitro [23–26]. This protein was termed “activin,” signifying its antagonistic effect to inhibin. The discovery of new genes related to the inhibin β subunit through the use of degenerate polymerase chain reaction (PCR) cloning methods further complicated the inhibinstory. There are now three known types of bioactive activins: activin A, activin AB, and activin B (Table 1). Three recently identified activins (C, D, and E) are not known to have any bioactivity and have a relatively restrictive tissue distribution. Activin has proven relevant to diverse research fields, including cell biology, developmental biology, and endocrinology. Activin bioassays have the same problems as inhibin, that is, a lack of specificity.

During purification of inhibin, another weak inhibitor of FSH secretion was identified, named follistatin, which was purified from both porcine [27] and bovine follicular fluid. It was soon realized that follistatin acted by binding to activin and thus neutralizing its actions. The protein is expressed abundantly in the granulosa cells of healthy antral follicles. Shimonaka et al. [27] demonstrated by double ligand blotting technique that activin A has two binding sites for follistatin, whereas inhibin A has one. From this, it can be shown that follistatin interacts with inhibin A and activin A through the common β subunit. Follistatin has the ability to neutralize activin-induced FSH release from pituitary cell cultures but this bioassay, like those for inhibins and activins, is susceptible to interference [28]. A number of immunoassays for total follistatin have now been established [29].

Another activin-binding protein of 70 amino acids (aa) has been identified as a follistatin-related gene (FLRG) product, based upon its primary sequence homology to follistatin and its modular architecture, which is remarkably similar to that of follistatin [30, 31]. Like follistatin, FLRG also interacts physically with activin A. This interaction prevents binding to activin receptors (ActRs) [31, 32], thus suggesting a regulatory role on activin-mediated cellular processes.

Both activin and inhibin have been classified as members of the transforming growth factor-β (TGF-β) superfamily [23,, 25,, 28,32–34]. They are synthesized as large precursors containing a signal sequence, a pro-domain of variable size, and a mature C-terminal segment that ranges from 110 to 140 aa in length. Within the mature segment, there are seven cysteine residues that are invariant across the...