Genetic Landscape and Diagnostic Perspectives in Azoospermia: Insights, Challenges, and Future Directions

Azoospermia, affecting 1% of men, can result from various factors: problems with the brain's hormone control center, issues with sperm production, or blockages in the reproductive tract. Genetic factors play a role in these categories, and genetic testing is standard for diagnosing azoospermia. The effectiveness of genetic tests varies by cause, being highest (90%) in Congenital Bilateral Absence of Vas Deferens and lowest (~30%) in Non-Obstructive Azoospermia caused by primary testicular failure. Whole-Exome Sequencing has revealed around 38 potential genes linked to Non-Obstructive Azoospermia. Understanding these genes could lead to better screening methods. Some genes are common in both male and female fertility issues, emphasizing the need for genetic guidance for female relatives of affected males.

Male infertility is a complex condition affecting 7–12% of men, with about 1% experiencing azoospermia, where no sperm is found in the ejaculate. There are three main causes: issues with the brain's control over hormones, problems in sperm production, and blockages in the reproductive tract. These can be linked to both acquired (such as infections or certain treatments) and congenital factors.

Azoospermia can be divided into two types: Obstructive Azoospermia, where the sperm-making process is unaffected due to a physical blockage, and Non-Obstructive Azoospermia, caused by testicular failure. Non-Obstructive Azoospermia can present in different ways in the testis tissue. Around 35–40% of azoospermia cases result from acquired conditions, while genetic factors play a role in various causes. Genetic testing is crucial for diagnosis, guiding treatment decisions, and providing appropriate counseling.

In clinical practice, routine tests like karyotype abnormalities and genetic screenings like Azoospermia Factor microdeletions are common for diagnosing azoospermic patients. Newer methods like Next Generation Sequencing (NGS) have revealed more genetic causes. This review aims to give an overview of the genetic factors involved in azoospermia.

Klinefelter Syndrome (47,XXY) is the most common genetic cause of Non-Obstructive Azoospermia (NOA). It's characterized by an extra X chromosome and can be challenging to diagnose due to varying symptoms. In newborn males, its prevalence is around 1 in 600, but among infertile men, it can affect 3–4%, rising to 10–12% in azoospermic individuals. Unfortunately, about 64% of patients are misdiagnosed or undiagnosed due to mild symptoms.

Klinefelter Syndrome has diverse clinical features including eunuchoid habitus, hypogonadism, gynecomastia, small testes, and neurocognitive deficits. It's often identified by a 47,XXY karyotype, but there are variations like higher-grade aneuploidies or mosaicisms. This extra X chromosome can come from errors during egg or sperm formation or early cell division after fertilization.

Regarding fertility, many with Klinefelter Syndrome face azoospermia due to testicular issues causing germ cell loss. However, some can have sporadic sperm production. Techniques like micro-Testicular Sperm Extraction (m-TESE) can retrieve sperm in some cases, enabling assisted reproduction methods like Intra-Cytoplasmic Sperm Injection (ICSI) with moderate success rates. The timing for better sperm retrieval success seems to be in late adolescence or early adulthood. Despite challenges, with the right approach, KS men can have biological children with no increased risk of Klinefelter Syndrome in their offspring.

Another anomaly causing azoospermia is the 46,XX testicular/ovo-testicular Disorder of Sex Development (DSD), also known as 46,XX male syndrome. It's a rare condition, where the presence or absence of the SRY gene determines the male phenotype. Those with the SRY gene usually have fully male genitalia but might face fertility issues due to lack of Y chromosome-linked genes essential for spermatogenesis. Sperm donation or adoption might be viable options for them to have children.

Additionally, the absence of the long arm of the Y chromosome (Yq−) and microdeletions of the Y Chromosome (AZF Deletions) are linked with azoospermia due to critical genes necessary for sperm production residing in these regions. AZF microdeletions are known to cause male infertility, and their occurrence is relatively frequent among those with idiopathic NOA. The deletions affect spermatogenesis-associated genes, making it challenging to pinpoint the exact genes responsible for the associated infertility.

These chromosomal anomalies and deletions illustrate the complex genetic factors involved in male infertility, posing challenges and opportunities for understanding and managing azoospermia.

The AZFa region is a segment of DNA about 792 kilobases long and holds two important genes: USP9Y and DDX3Y. USP9Y creates a protein that helps regulate protein turnover, and its absence can lead to various testicular issues, from low sperm production to normal levels. Meanwhile, DDX3Y is responsible for producing an RNA helicase and plays a role in early sperm development. If DDX3Y is missing due to AZFa deletion, it results in a condition called SCOS, characterized by no germ cells in the testis, small testes, and high levels of FSH.

The AZFb deletion removes a DNA segment of about 6.2 megabases, impacting around 32 copies of genes and other units that are crucial for the maturation of sperm cells. This deletion leads to a halt in germ cell development at the spermatocyte stage, causing azoospermia. This condition might look like obstructive azoospermia, as testicular volume and hormone levels can appear normal.

AZFc involves a set of 12 genes present in variable numbers of copies, resulting in a total of 32 copies. Complete AZFc deletion leads to various clinical outcomes, sometimes allowing the detection of a small number of sperm in ejaculate, though typically less than 2 million/mL. Given a gradual decrease in sperm count, sperm preservation is recommended. In cases of azoospermia, the testis might show a range of conditions from SCOS to low sperm production.

Testing for Y chromosome deletions follows standardized procedures outlined by EAA/EMQN guidelines. However, specific variations in the genetic markers used in these guidelines, like sY84, might be more frequent in certain populations than others. It's essential to consider alternatives if these markers fail to amplify during testing. Detecting AZF deletions not only helps diagnose but also predicts the success of certain sperm retrieval procedures like TESE.

Regarding AZFa and AZFb deletions, complete deletions in these regions significantly decrease the chances of finding sperm in the testes. However, in some cases, partial deletions may allow for residual spermatogenesis. The identification of specific markers, like sY1192, has become crucial for deciding whether TESE might be attempted.

For AZFc microdeletion carriers, sperm retrieval from testicles through m-TESE shows a success rate of 50–60%. These individuals might produce a significant portion of sperm with nullisomy for sex chromosomes. Yq microdeletions might lead to Y chromosomal instability and, in some cases, the formation of 45,X0 cell lines. This mosaicism can affect the success of sperm retrieval.

Moving on to monogenic forms of azoospermia, various genetic defects contribute to primary testicular failure. Though not yet a routine part of diagnosing NOA, advancements in sequencing techniques have identified several candidate genes, with around 17 validated through multiple studies. These genes play a role in spermatogenesis and could account for up to 70% of NOA cases without acquired causes, termed idiopathic NOA (iNOA). This understanding is rapidly growing due to next-generation sequencing, revealing novel candidate genes responsible for primary testicular failure.

In conclusion, the landscape of azoospermia presents a complex interplay of genetic, acquired, and congenital factors, contributing to the varied etiology of male infertility. Genetic testing has emerged as a critical tool for diagnosis, offering insights into the diverse genetic anomalies associated with different forms of azoospermia. The advancements in genetic analysis, particularly through Next Generation Sequencing (NGS), have unveiled a multitude of candidate genes and chromosomal regions linked to Non-Obstructive Azoospermia (NOA). Conditions like Klinefelter Syndrome, AZF deletions, and AZF microdeletions showcase the pivotal role of specific genes in spermatogenesis and fertility. However, challenges persist in diagnosing and managing these conditions, as some cases remain undiagnosed due to mild symptoms or complexities in genetic variations. As research progresses, understanding these genetic underpinnings not only aids in accurate diagnosis but also opens avenues for tailored treatments, assisted reproductive techniques, and potential interventions for male infertility, offering hope for individuals affected by azoospermia and paving the way for enhanced genetic counseling and precision medicine in this domain.