Androstenedione (4-androstene-3,17-dione) is a 19-carbon steroid hormone produced by the adrenal glands, gonads, and placenta. It acts as a direct precursor in the biosynthesis of testosterone and estrone, bridging the metabolic pathways of both androgens and estrogens.

First identified in the early 20th century, androstenedione has since been implicated in a broad spectrum of physiological processes, including sexual differentiation, reproductive health, and metabolic regulation [1,2].

As a biomarker, androstenedione holds clinical significance in evaluating conditions of androgen excess, such as polycystic ovary syndrome (PCOS), congenital adrenal hyperplasia (CAH), and various adrenal or gonadal neoplasms. Elevated levels in blood or saliva may indicate enzymatic blockages in steroidogenesis, hyperfunctioning adrenal tissue, or peripheral conversion anomalies. Accurate quantification of androstenedione is therefore essential for endocrine diagnosis, treatment monitoring, and understanding of disease pathophysiology [3].

This review delves into the diagnostic utility of androstenedione, the challenges posed in its immunodetection, and novel strategies to enhance analytical specificity through monoclonal antibody development.

Androstenedione is synthesized from 17-hydroxyprogesterone or dehydroepiandrosterone (DHEA) through enzymatic action involving 17,20-lyase and 3β-hydroxysteroid dehydrogenase. In males, the primary site of production is the Leydig cells of the testes, while in females, the adrenal cortex and ovarian theca cells contribute significantly [4].

Once produced, androstenedione can be converted into testosterone via 17β-hydroxysteroid dehydrogenase or into estrone through aromatase activity. This dual conversion pathway makes androstenedione a pivotal node in the steroidogenic cascade. Circulating levels are influenced by age, sex, circadian rhythms, and physiological states such as pregnancy or menopause [5].

PCOS affects up to 10% of women of reproductive age and is characterized by hyperandrogenism, chronic anovulation, and polycystic ovarian morphology. Androstenedione, alongside testosterone, is often elevated in PCOS and reflects ovarian and adrenal androgen production [6]. Since it is less bound by sex hormone-binding globulin (SHBG) compared to testosterone, its measurement may offer a more accurate reflection of bioavailable androgen levels.

CAH is a group of autosomal recessive disorders resulting from enzyme deficiencies in the steroidogenic pathway, most commonly 21-hydroxylase deficiency. This defect leads to cortisol deficiency and overproduction of ACTH, driving excessive androstenedione synthesis. Elevated androstenedione is a hallmark of the disease and serves as a key marker for both diagnosis and therapeutic monitoring [7,8].

Neoplastic lesions in adrenal or gonadal tissues may autonomously produce androstenedione. In such cases, its elevation is often disproportionate to other steroids, providing a biochemical fingerprint that can assist in tumor localization and characterization [9].

In males, low androstenedione levels may indicate impaired testicular function, pituitary insufficiency, or late-onset hypogonadism. Conversely, in females, low levels are less commonly of diagnostic concern but may arise in primary adrenal insufficiency [10].

Gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) provide superior analytical specificity and sensitivity. These methods allow for the separation of closely related steroids prior to detection, minimizing cross-reactivity. Despite their advantages, the high cost, technical complexity, and need for extensive sample preparation limit their routine clinical application [13,14].

Historically, radioimmunoassays (RIAs) and enzyme-linked immunosorbent assays (ELISAs) have been employed for androstenedione measurement due to their simplicity and high throughput. However, these assays suffer from limited specificity due to cross-reactivity with structurally similar steroids, particularly testosterone, DHEA, and 17-hydroxyprogesterone [11,12].

In fact, steroids are low-molecular-weight, non-immunogenic molecules (haptens) with a common cyclopentanoperhydrophenanthrene core. This shared structure among steroids such as androstenedione, testosterone, and estrone creates a significant challenge in eliciting antibodies with absolute specificity [15].

Cross-reactivity arises when antibodies raised against androstenedione also recognize similar epitopes on related steroids. This is especially problematic in immunoassays, where such cross-binding can inflate or confound the true concentration of the analyte of interest [16].

This is why SYnAbs has developed highly specific monoclonal antibodies to androstenedione without cross-reactivity to testosterone, DHEA, and 17-hydroxyprogesterone.

To generate an immune response, androstenedione must be conjugated to a carrier protein such as bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH). The site of conjugation is critical: if the linkage occurs at a position that is structurally conserved across steroid analogs, the resulting antibodies may be broadly reactive [17].

Optimizing hapten design to expose unique moieties of androstenedione while masking conserved regions can improve antibody specificity. However, this requires precise chemical modification and an understanding of epitope-antibody interactions at the molecular level [18].

Androstenedione serves as a crucial biomarker in the diagnosis and management of various endocrine disorders. Despite its clinical utility, the accurate immunodetection of androstenedione is limited by its structural similarity to other steroid hormones. The development of highly specific monoclonal antibodies was a formidable challenge for SYnAbs team.

SYnAbs hapten design expertise, its unique rat-LOU species also with technologies to overcome immunotolerance, and screening technologies have kept their promises for improving assay specificity and robustness thanks to the generation of innovative monoclonal antibodies. Complementary methods such as LC-MS/MS will continue to play a role in confirming potential ambiguous results and benchmarking immunoassay performance.

As precision medicine evolves, the ability to accurately and selectively quantify hormones like androstenedione will be integral to personalized diagnostics and targeted therapies. That's why SYnAbs has made the generation of ultra-specific monoclonal antibodies to steroids one of its specialty and will continue to investigate the field of possibilities.

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