What investigations are recommended for a patient presenting with azoospermia (absence of sperm)?

Investigations for Azoospermia

The evaluation of azoospermia requires confirmatory semen analyses, hormonal assessment (FSH and testosterone), physical examination focusing on testicular size and vas deferens presence, and genetic testing (karyotype and Y-chromosome microdeletion analysis) to distinguish obstructive from non-obstructive causes. 1

Confirmatory Semen Analysis

• Perform at least two semen analyses at least one month apart to confirm true azoospermia, as approximately 18-23% of men initially diagnosed with azoospermia will have rare sperm identified on pellet examination after centrifugation 1
• Patients should abstain from sexual activity for 2-3 days before collection 1
• Specimens must be kept at room or body temperature during transport and examined within one hour of collection 1
• The laboratory must centrifuge the ejaculate and examine the pellet under microscopy for rare sperm, as this can identify motile or non-motile sperm in nearly one-quarter of cases 1

Physical Examination

Assess testicular size and consistency as the primary physical finding: normal-sized testes (>15 mL or >4 cm length) suggest obstructive azoospermia, while atrophic testes indicate non-obstructive azoospermia 1, 2

• Palpate for presence of vasa deferentia bilaterally, as congenital bilateral absence can be diagnosed by physical examination and indicates obstructive azoospermia 1
• Examine for varicocele, hydrocele, or other scrotal abnormalities 1
• Assess epididymal consistency: dilated and/or indurated epididymides suggest obstructive azoospermia 1
• Evaluate secondary sex characteristics including hair distribution and breast development 1
• Perform digital rectal examination to assess prostate size and consistency 1

Hormonal Evaluation

Measure serum FSH and testosterone as the primary hormonal assessment to distinguish obstructive from non-obstructive azoospermia 1, 3

• FSH >7.6 IU/L typically indicates non-obstructive azoospermia (spermatogenic failure), while normal FSH suggests obstructive azoospermia 1, 2
• Measure luteinizing hormone (LH) as part of the basic hormonal workup to assess for hypogonadotropic hypogonadism 1
• Consider anti-Müllerian hormone (AMH) testing, as lower levels may predict better sperm retrieval outcomes 1

Critical caveat: FSH levels alone cannot definitively predict sperm retrieval success—up to 50% of men with non-obstructive azoospermia and elevated FSH still have retrievable sperm with testicular sperm extraction 1, 2

Distinguishing Obstructive vs. Non-Obstructive Azoospermia

Obstructive Azoospermia Pattern:

• Normal-sized testes (>15 mL) 1
• Normal FSH levels (<7.6 IU/L) 1, 2
• Dilated and/or indurated epididymides 1
• Possible absence of vas deferens 1
• Low ejaculate volume (<1.5 mL) may suggest ejaculatory duct obstruction or congenital bilateral absence of vas deferens 1

Non-Obstructive Azoospermia Pattern:

• Atrophic testes 1, 2
• Elevated FSH (>7.6 IU/L) 1, 2
• Normal epididymides and vas deferens 1
• Normal semen volume and pH 2

Genetic Testing

Karyotype testing is mandatory for all patients with azoospermia or severe oligospermia (<5 million/mL) to identify chromosomal abnormalities including Klinefelter syndrome 1, 4, 3

Y-chromosome microdeletion analysis is mandatory for azoospermia or sperm concentration <1 million/mL, testing specifically for AZFa, AZFb, and AZFc regions 1, 3, 5

• Complete AZFa and AZFb deletions result in almost zero likelihood of sperm retrieval 2
• CFTR gene testing should be offered to female partners of men with congenital bilateral absence of vas deferens, as these men have a high risk of being cystic fibrosis carriers 1, 3

Additional Specialized Testing

Post-Ejaculatory Urinalysis

• Indicated when ejaculate volume is <1 mL (except in patients with bilateral vasal agenesis or hypogonadism) to diagnose retrograde ejaculation 1

Transrectal Ultrasonography (TRUS)

• Indicated in azoospermic patients with palpable vasa and low ejaculate volumes to evaluate for ejaculatory duct obstruction 1

Scrotal Ultrasonography

• Indicated when physical examination of the scrotum is difficult or inadequate 1
• Indicated when a testicular mass is suspected 1
• Useful for testicular volume assessment, detection of testicular tumors, and assessment of testicular architecture 1
• Non-homogeneous testicular architecture suggests testicular dysgenesis and impaired spermatogenesis in non-obstructive azoospermia 1
• Testicular microcalcifications are associated with 18-fold higher prevalence of testicular cancer in infertile men 1

Critical Pitfalls to Avoid

• Never prescribe testosterone replacement therapy to men desiring fertility, as it suppresses spermatogenesis through negative feedback and can cause azoospermia 1, 2
• Do not rely on a single semen analysis—biological variability requires confirmation 1
• FSH levels cannot definitively exclude the possibility of sperm retrieval, as men with maturation arrest can have normal FSH despite severe spermatogenic dysfunction 1
• Before proceeding with intracytoplasmic sperm injection (ICSI), patients must be informed about potential genetic abnormalities associated with azoospermia and their implications for offspring 1