Testosterone, Androgens, and Andrological Therapies: A Comprehensive Overview

Molecular Mechanisms and Biotechnological Approaches in Androgen Synthesis

Testosterone (TS) and its derivative boldenone (BD) can be produced from natural plant sterols through a series of enzymatic reactions known as cascade biotransformations. In such processes, specific microbial strains such as Mycolicibacterium neoaurum convert phytosterols into primary androstane steroids like androstenedione (AD) or androstadienedione (ADD). Subsequently, a filamentous fungus, for example a strain of Curvularia sp., with high 17β-hydroxysteroid dehydrogenase (17β-HSD) activity catalyzes the reduction of the 17-carbonyl group of AD or ADD, yielding TS or BD with high efficiency. Optimization studies indicate that when using 60 g/L (dry weight) of fungal biomass and adding a solubilizing agent such as methylated β-cyclodextrin (MCD) at a molar ratio of 0.5:1 (MCD:steroid), the yield of the desired product can be maximized. Under these conditions, AD at 2.48 g/L can be converted to testosterone with conversion rates exceeding 90% in as little as 6 hours, whereas the reduction of ADD to boldenone may also achieve yields as high as 97% under lower substrate concentrations (1–2 g/L) within a similar timeframe. Longer incubation times or higher substrate concentrations generally reduce product yield due to factors such as limited substrate solubility and possible toxicity to fungal cells. These biocatalytic strategies obviate the need for complex chemical synthesis, thus providing a greener process using renewable raw materials and milder conditions [1].

Below is a representative data table summarizing the cascade biotransformation process:

Table 1. Cascade biotransformation efficiency in the production of testosterone and boldenone from phytosterol.

The fungal genome analysis of Curvularia sp. using transcriptomic data revealed the presence of six candidate genes encoding 17β-HSD enzymes. These genes, which share 28–57% nucleotide homology with known 17β-HSD sequences, are constitutively expressed in the fungal mycelium. Differential gene expression studies suggested that while 17β-HSD activity is maintained consistently, inducible cytochrome P450 monooxygenase activities can mediate additional modifications such as 7α-hydroxylation of testosterone. Notably, protein synthesis inhibitors like cycloheximide can be applied during the biotransformation process to suppress the inducible hydroxylase, thereby preserving testosterone yields and minimizing unwanted side-product formation [1].

Clinical Applications of Testosterone Replacement and Andrological Therapies

Testosterone replacement therapy (TRT) is widely applied in cases of hypogonadism and other androgen deficiency syndromes. Congenital hypogonadotropic hypogonadism, a disorder resulting from complex genetic anomalies—including oligogenic inheritance involving mutations in the FGFR1 and GNRHR genes—can lead to delayed or absent pubertal development and subsequent infertility. Genetic investigations using next-generation sequencing in affected individuals have identified novel pathogenic variants in these genes. For instance, a recent study reported a patient with heterozygous variants in FGFR1 and GNRHR, leading to normosmic hypogonadotropic hypogonadism along with secondary osteoporosis. Comprehensive evaluation in such cases involves not only clinical and biochemical assessments but also detailed genetic testing to inform personalized therapeutic strategies [2].

Treatment guidelines now advocate for early intervention in pediatric and adolescent patients when there is evidence of pubertal delay. In cases of delayed puberty, a “wait and see” approach may be implemented initially; however, if there is no progress within six months, low-dose testosterone therapy may be initiated. Low-dose TRT administered via intramuscular injections or transdermal applications has been shown to induce secondary sexual characteristics, promote growth velocity, and improve overall quality of life without adverse effects on final adult height. Moreover, multidisciplinary guidelines developed by the Italian Society of Andrology and Sexual Medicine in collaboration with the Italian Society for Pediatric Endocrinology and Diabetology emphasize that andrological disorders often have origins in childhood, necessitating early diagnosis and timely management to prevent long-term complications, including infertility and metabolic derangements [3].

In clinical practice, the monitoring of biochemical markers such as serum luteinizing hormone (LH), follicle-stimulating hormone (FSH), inhibin B, and testosterone levels is central to optimizing treatment outcomes. Moreover, imaging modalities including scrotal ultrasonography are increasingly used for accurate assessment of testicular volume and to detect associated pathologies like varicocele. Recent evidence also supports the use of normative data in evaluating testicular asymmetry and growth, which are essential for forecasting future fertility potential [3].

Impact of Androgen Therapies on Cardiovascular and Neurodevelopmental Outcomes

The cardiovascular system and neurodevelopment are two critical domains influenced by androgen levels. In transgender individuals undergoing GAHT, studies have revealed that exogenous androgen or estrogen treatment results in cardiac structural and electrocardiographic alterations. AI-driven ECG algorithms have been recently developed to distinguish male from female cardiac electrical patterns with over 90% accuracy. In one study, transgender women receiving feminizing GAHT exhibited a significant decrease in the male pattern probability on ECG, along with prolongation of the QTc interval and PR interval changes consistent with the hormonal milieu. Conversely, transgender men receiving masculinizing therapy showed increases in AI-predicted male electrocardiographic features and QTc shortening. These findings are essential as they highlight that GAHT not only affects reproductive tissues but may also alter cardiac ion channel expression and myocardial physiology, thereby potentially impacting cardiovascular risk profiles [4].

Neurodevelopmental outcomes in specific populations such as infants with 47,XXY (Klinefelter syndrome) have also been investigated. A double-blind randomized controlled trial (the TESTO trial) examined the effects of testosterone injections administered during the mini-puberty period in infants with non-mosaic 47,XXY. The trial reported that exogenous testosterone led to measurable physical changes, including improved lean mass accumulation and reduced percent fat mass, while effectively suppressing endogenous gonadotropins and inhibin B. However, short-term neurodevelopmental outcomes measured by standardized motor scales did not differ significantly between testosterone-treated and placebo groups. These findings suggest that while testosterone treatment in early infancy may have beneficial effects on body composition, its role in modulating neurodevelopment remains uncertain and warrants longer-term investigations [4].

The integration of these findings into clinical practice requires careful assessment of both benefits and potential risks. Suppression of the hypothalamic-pituitary-gonadal (HPG) axis in infancy as a side effect of exogenous testosterone treatment, as well as its impact on long-term fertility and metabolic health, must be considered during therapeutic decision-making. In this context, long-term follow-up studies are crucial to ascertain the durability of physical changes and to assess subsequent fertility, neurocognitive, and cardiovascular outcomes [4].

Advances in Diagnostic and Monitoring Technologies

Recent technological innovations have enhanced the precision and efficacy of andrological therapies. AI-based analysis of ECGs represents one such breakthrough, where algorithms trained on large datasets can provide objective quantification of subtle electrocardiographic changes induced by hormone therapy. These AI tools complement traditional methods of interval measurement and can help personalize treatment by identifying individuals at risk for adverse cardiovascular events.

In addition to AI, advanced imaging modalities and biochemical assessments now allow clinicians to monitor treatment response in real time. For example, scrotal ultrasound imaging is used to assess testicular volume and vascular flow in adolescents with varicocele, while air displacement plethysmography (PEAPOD) offers precise body composition analysis in infants undergoing hormone therapy. Furthermore, genomic studies using next-generation sequencing have revolutionized the diagnosis of congenital hypogonadism, enabling the detection of pathogenic variants in critical genes like FGFR1 and GNRHR. These diagnostic advances provide a more holistic approach to andrological care, ensuring that therapies are tailored to individual genetic, biochemical, and physiological profiles [2, 3].

Future Perspectives and Clinical Implications

The ongoing refinement of biotechnological processes for steroid synthesis, including cascade biotransformations mediated by actinobacteria and mold fungi, not only promises more sustainable production methods for testosterone and boldenone but may also pave the way for the creation of novel steroid derivatives with custom-tailored pharmacological properties. With growing evidence supporting the use of exogenous testosterone during critical developmental windows, future research should focus on the long-term safety and efficacy of such interventions, particularly in populations with sex chromosome aneuploidies like 47,XXY.

Additionally, further incorporation of AI and machine learning models into clinical practice may refine risk stratification and therapeutic monitoring, thereby optimizing outcomes in transgender care and adolescent endocrinology. The evolution of clinical guidelines that integrate these technologies, combined with advances in molecular genetics, is likely to transform andrological therapies into an era of personalized medicine. As research continues, it is essential that clinicians remain updated on emerging data and incorporate multidisciplinary perspectives in managing complex endocrine and reproductive disorders [3, 4].

Conclusion

Testosterone and androgen therapies sit at the nexus of molecular biology, biotechnology, and clinical medicine. Advanced biotransformation techniques now allow for the green production of testosterone and boldenone from natural phytosterols, while genetics and AI are redefining the diagnosis and management of andrological disorders. Clinical applications of these therapies extend from treating congenital hypogonadism and pubertal delay to refining GAHT protocols in transgender individuals. Although short-term outcomes in body composition and electrocardiographic parameters have been promising, especially in infants with 47,XXY and transgender populations, larger long-term studies are necessary to validate the safety, neurodevelopmental impact, and metabolic consequences of these interventions. In the context of evolving multidisciplinary guidelines, the combination of advanced diagnostics, precise biotechnological methods, and innovative monitoring strategies heralds a new era in personalized andrological care.

Frequently Asked Questions

What are androgens and why are they important?
Androgens are steroid hormones, with testosterone being the most prominent, that play key roles in driving male sexual differentiation, the development of secondary sexual characteristics, and maintaining muscle mass and bone density. They also influence mood, cognitive function, and overall metabolic health.

How are testosterone and its derivatives produced biotechnologically?
Modern biotechnological methods use cascade biotransformations whereby phytosterols are first converted to androstane derivatives by bacteria (such as Mycolicibacterium neoaurum), followed by reduction by fungal enzymes (e.g., 17β-HSD from Curvularia sp.) to yield testosterone or boldenone. Optimized conditions including appropriate substrate concentrations and the use of cyclodextrins improve product yield.

What is the significance of genetic testing in hypogonadotropic hypogonadism?
Genetic testing helps identify pathogenic variants in genes such as FGFR1 and GNRHR, which are central to the development of the hypothalamic-pituitary-gonadal axis. Discovering these variants can guide personalized treatment plans, assist in family counseling, and inform long-term management strategies.

How does testosterone therapy affect cardiovascular outcomes?
Testosterone therapy can modulate myocardial ion channels and influence electrocardiographic patterns, such as changes in the QTc and PR intervals. AI-based ECG analysis has demonstrated that hormone therapy induces gender-congruent changes in the cardiac electrical profile. However, the long-term cardiovascular impact needs further investigation.

What are the potential benefits of early testosterone treatment in infants with 47,XXY?
Early testosterone treatment in infants with 47,XXY (Klinefelter syndrome) may improve body composition by reducing fat mass and increasing lean mass, although studies have not shown significant short-term neurodevelopmental improvements. The therapy may also suppress gonadotropins, but routine treatment is not currently recommended without further long-term dat

References

1. Kollerov, V. V., Timakova, T. A., Shutov, A. A., Donova, M. V., & Rosa, E. A. R. (2024). Boldenone and testosterone production from phytosterol via one-pot cascade biotransformations. Journal of Fungi, 10(12), 830. https://doi.org/10.3390/jof10120830

2. Davis, S. M. D., Howell, S. M., Janusz, J., Lahlou, N., Reynolds, R. M. D., Thompson, T., Swenson, K., Wilson, R. P., Ross, J., Zeitler, P., & Tartaglia, N. (2024). Testosterone effects on short-term physical, hormonal, and neurodevelopmental outcomes in infants with 47,XXY/Klinefelter syndrome: The TESTO randomized controlled trial. medRxiv. https://doi.org/10.1101/2024.12.09.24318726

3. Bonomi, M., Rochira, V., Pasquali, D., Balercia, G., Jannini, E. A., Ferlin, A., … & Klinefelter ItaliaN Group. (2024). “Management of andrological disorders from childhood and adolescence to transition age: Guidelines from the Italian Society of Andrology and Sexual Medicine (SIAMS) in collaboration with the Italian Society for Pediatric Endocrinology and Diabetology (SIEDP)—Part-1.” Journal of Endocrinological Investigation. https://doi.org/10.1007/s40618-024-02435-x

4. Davis, S. M. D., Howell, S. M., Janusz, J., Lahlou, N., Reynolds, R. M. D., Thompson, T., Swenson, K., Wilson, R., Ross, J., Zeitler, P., & Tartaglia, N. (2024). Artificial intelligence evaluation of electrocardiographic characteristics and interval changes in transgender patients on gender-affirming hormone therapy. Journal of Endocrinological Investigation. https://pubmed.ncbi.nlm.nih.gov/11750187/

5. (No author listed). (n.d.). Congenital hypogonadotropic hypogonadism with novel pathogenic variants in FGFR1 and GNRHR. PubMed. https://pubmed.ncbi.nlm.nih.gov/11733946/

6. (No author listed). (n.d.). Testosterone effects on short-term physical, hormonal, and neurodevelopmental outcomes in infants with 47,XXY/Klinefelter syndrome: The TESTO randomized controlled trial. medRxiv. https://doi.org/10.1101/2024.12.09.24318726