Chemical Testing

For the vast majority of fertility and post-vasectomy cases, the foundational routine tests - physical examination and microscopic analysis - provide all the information we need

However, in certain complex cases - particularly in the workup of an azoospermic man (a man with no sperm in his ejaculate) - we need to move from general assessment to specific investigation. We need to become detectives. The question is no longer “Is there a problem?” but rather, “Where is the problem?”

Is the “sperm factory” (the testes) shut down? Or is the factory working fine, but there’s a blockage in the “plumbing” somewhere along the male reproductive tract?

This is where chemical testing comes in. These are not routine tests. They are second-line, specialized assays that we use as biochemical markers to functionally map the reproductive tract and pinpoint the site of an obstruction or dysfunction. Each chemical analyte we measure is a specific product of a particular gland. Its presence or absence tells us if that gland is functioning and if its ducts are open

Fructose: The Seminal Vesicle Function Test

This is the most important and most frequently performed chemical test on semen. It is a direct marker of seminal vesicle function and ejaculatory duct patency

Physiology Recap
- Source: Fructose is produced almost exclusively by the seminal vesicles.
- Function: It is the primary energy source for sperm motility
- The seminal vesicles contribute ~60-70% of the total semen volume, and this fluid is alkaline
When Do We Order This Test? (The Clinical Trigger)
- We order a fructose test when we see a specific constellation of findings from the basic analysis:
  1. Azoospermia: No sperm are seen on the microscopic exam
  2. AND Low Ejaculate Volume: The volume is < 1.5 mL
- This combination suggests that the largest contributor to volume - the seminal vesicles - may be absent, dysfunctional, or blocked
Methodology
- The test is typically a simple, qualitative colorimetric screening test using a resorcinol reagent.
- When heated with fructose, the resorcinol reagent produces a red-orange color
- Quantitative spectrophotometric methods also exist
- Normal: The test is positive (a color change occurs), indicating the presence of fructose
- Abnormal: The test is negative (no color change), indicating the absence of fructose
Interpretation & Diagnostic Significance
- Normal Fructose (Positive Test)
  - This tells us two things: 1) The seminal vesicles are present and producing fructose, and 2) The ejaculatory duct is open (patent)
  - If the patient is still azoospermic, this result points strongly toward a testicular problem (non-obstructive azoospermia).: The plumbing is open, but the factory isn’t producing any sperm. This is often due to primary testicular failure
- Absent Fructose (Negative Test)
  - This is a critical finding that, combined with low volume and acidic pH, pinpoints the problem to the very end of the reproductive tract
  - It indicates either:
    1. Ejaculatory Duct Obstruction (EDO): The seminal vesicles are working, but a blockage prevents their secretions from entering the ejaculate
    2. Congenital Bilateral Absence of the Vas Deferens (CBAVD): A genetic condition (often associated with cystic fibrosis) where the vas deferens and, in most cases, the seminal vesicles, never developed
  - This result confirms a diagnosis of obstructive azoospermia.

Neutral Alpha-Glucosidase (NAG): The Epididymal Patency Test

This is a more specific marker used to investigate obstructions that are “upstream” from the seminal vesicles

Physiology Recap
- Source: NAG is an enzyme produced specifically in the epididymis.
- Function: The epididymis is where sperm mature and acquire motility
When Do We Order This Test? (The Clinical Trigger)
- The trigger for NAG is different from fructose. We order it when we find:
  1. Azoospermia
  2. AND Normal Ejaculate Volume (≥ 1.5 mL)
  3. AND Normal Fructose
- This pattern tells us that the seminal vesicles and prostate are working fine (hence the normal volume and fructose). Therefore, if there’s an obstruction, it must be higher up - in the epididymis or vas deferens
Methodology
- A spectrophotometric assay that measures the enzymatic activity of NAG
Interpretation & Diagnostic Significance
- Normal NAG Level
  - This indicates that the epididymis is functional and patent
  - If the patient is still azoospermic, this finding, like a normal fructose test, points strongly to primary testicular failure (non-obstructive azoospermia).
- Low NAG Level
  - This is a highly specific finding that strongly suggests an obstruction within the epididymis.
  - This is the classic profile of obstructive azoospermia: at the epididymal level. The testes are making sperm (which is why the epididymis is stimulated to make NAG), but a blockage prevents both the sperm and the NAG from getting into the ejaculate. This is crucial information for a surgeon who may be able to repair the obstruction

Prostate Gland Markers: Zinc, Citric Acid, Acid Phosphatase

These analytes are used to assess the secretory function of the prostate gland

Physiology Recap
- Source: These substances are all produced and concentrated in the prostate gland.
- Function: They contribute to the chemical environment of the semen, with acid phosphatase and PSA being key for liquefaction
When Do We Order These Tests?
- When there is evidence of prostatic dysfunction, such as:
  - Failure of liquefaction or hyperviscosity.
  - Abnormal pH
  - Low semen volume where a seminal vesicle issue has been ruled out
Interpretation
- Decreased levels of zinc, citric acid, and acid phosphatase are indicative of a prostatic secretory dysfunction.: This can be caused by inflammation (prostatitis) or other glandular disorders

Synthesis: Diagnostic Algorithms with Chemical Markers

Let’s put it all together. An azoospermic patient comes to the clinic. Here is how we use chemical markers to find the cause:

Scenario 1: Ejaculatory Duct Obstruction / CBAVD
- Basic Semen Analysis Findings
  - Azoospermia
  - Low Volume (< 1.5 mL)
  - Acidic pH (< 7.2)
- Chemical Marker Profile
  - Fructose: Absent
- Most Likely Diagnosis: Ejaculatory Duct Obstruction or Congenital Bilateral Absence of the Vas Deferens (CBAVD)
- Type of Azoospermia: Obstructive
Scenario 2: Epididymal Obstruction
- Basic Semen Analysis Findings
  - Azoospermia
  - Normal Volume (≥ 1.5 mL)
  - Normal pH
- Chemical Marker Profile
  - Fructose: Present
  - Neutral Alpha-Glucosidase (NAG): Low
- Most Likely Diagnosis: Epididymal Obstruction
- Type of Azoospermia: Obstructive
Scenario 3: Primary Testicular Failure
- Basic Semen Analysis Findings
  - Azoospermia
  - Normal Volume (≥ 1.5 mL)
  - Normal pH
- Chemical Marker Profile
  - Fructose: Present
  - Neutral Alpha-Glucosidase (NAG): Normal
- Most Likely Diagnosis: Primary Testicular Failure
- Type of Azoospermia: Non-Obstructive

Conclusion

Chemical testing transforms semen analysis from a descriptive science into a precise diagnostic tool. It allows us to functionally dissect the male reproductive tract without invasive procedures. By intelligently selecting these tests based on the initial semen analysis results, we can provide the urologist or reproductive specialist with a clear roadmap, distinguishing between a treatable obstruction and a more complex issue of testicular failure. This is laboratory science at its most consultative

Physical Testing

Microscopic Analysis