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Clinical Pathology

Semen Analysis for Investigation of Infertility

By Dayyal Dg.Twitter Profile | Updated: Saturday, 16 December 2023 21:41 UTC
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Free vector infertility diagnosis concept illustration.
Free vector infertility diagnosis concept illustration. Freepik / @storyset

Semen, also known as seminal fluid, is a complex fluid discharged from the male genital tract, containing spermatozoa capable of fertilizing female ova. The intricate process of semen production involves several key structures:

  1. Testes: Male gametes, or spermatozoa, are generated within the testes, constituting 2-5% of the total semen volume.
  2. Epididymis: Following their production in the testes, spermatozoa undergo maturation and storage in the epididymis. This structure secretes essential components such as potassium, sodium, and glycerylphosphorylcholine—an energy source for sperms.
  3. Vas Deferens: Spermatozoa travel through the vas deferens to reach the ampulla, another storage site. The ampulla contributes ergothioneine, a yellowish fluid with chemical-reducing properties, and fructose, serving as a nutritional source for spermatozoa.
  4. Seminal Vesicles: During ejaculation, seminal vesicles and the prostate contribute nutritive and lubricating fluids. Seminal vesicles produce a fluid rich in fructose, amino acids, citric acid, phosphorous, potassium, and prostaglandins, contributing 50% to the overall semen volume.
  5. Prostate: Approximately 40% of the semen volume is attributed to prostatic secretions. These secretions contain citric acid, acid phosphatase, calcium, sodium, zinc, potassium, proteolytic enzymes, and fibrolysin.
  6. Bulbourethral Glands of Cowper: These glands secrete mucus, enhancing the lubrication and overall function of the semen.
Box 1: Contributions to semen volume
  • Testes and epididymis: 10%
  • Seminal vesicles: 50%
  • Prostate: 40%
  • Cowper’s glands: Small volume

Table 1 and Table 2 display the standard reference values for semen analysis.

Table 1: Normal values of semen analysis (World Health Organization, 1999)
TestResult
Volume ≥2 ml
pH 7.2 to 8.0
Sperm concentration ≥20 million/ml
Total sperm count per ejaculate ≥40 million
Morphology ≥30% sperms with normal morphology
Vitality ≥75% live
White blood cells <1 million/ml
Motility within 1 hour of ejaculation
  • Class A: ≥25% rapidly progressive
  • Class A and B: ≥50% progressive
Mixed antiglobuiln reaction (MAR) test <50% motile sperms with adherent particles
Immunobead test <50% motile sperms with adherent particles
Table 2: Biochemical variables of semen analysis (World Helath Organization, 1992)
Total fructose (seminal vesicle marker) ≥13 μmol/ejaculate
Total zinc (Prostate marker) ≥2.4 μmol/ejaculate
Total acid phosphatase (Prostate marker) ≥200U/ejaculate
Total citric acid (Prostate marker) ≥52 μmol/ejaculate
α-glucosidase (Epididymis marker) ≥20 mU/ejaculate
Carnitine (Epididymis marker) 0.8-2.9 μmol/ejaculate

Indications for Semen Analysis

Box 2: Tests done on seminal fluid
  • Physical examination: Time to liquefaction, viscosity, volume, pH, color
  • Microscopic examination: Sperm count, vitality, motility, morphology, and proportion of white cells
  • Immunologic analysis: Antisperm antibodies (SpermMAR test, Immunobead test)
  • Bacteriologic analysis: Detection of infection
  • Biochemical analysis: Fructose, zinc, acid phosphatase, carnitine.
  • Sperm function tests: Postcoital test, cervical mucus penetration test, Hamster egg penetration assay, hypoosmotic swelling of flagella, and computer-assisted semen analysis

The availability of semen for examination provides a unique opportunity for the direct assessment of male germ cells, a capability not paralleled in the examination of female germ cells. Conducting a semen analysis demands a high level of skill and ideally should be carried out in a specialized andrology laboratory.

  1. Investigation of Infertility: Semen analysis serves as the initial crucial step in exploring cases of infertility. Approximately 30% of infertility cases stem from male-related issues.
  2. Assessment of Vasectomy Effectiveness: Semen analysis is employed to verify the success of vasectomy by confirming the absence of sperm, ensuring the desired outcome of the procedure.
  3. Verification or Refutation of Sterility-Related Paternity Denials: Semen analysis plays a pivotal role in supporting or refuting claims of paternity denial based on alleged sterility, contributing valuable insights to legal matters.
  4. Examination of Medicolegal Cases: Semen analysis is instrumental in examining vaginal secretions or clothing stains for the presence of semen in medicolegal cases, aiding in the determination of crucial forensic evidence.
  5. Donor Selection for Artificial Insemination: Semen analysis is integral in the selection of donors for artificial insemination, ensuring the quality and viability of sperm for successful reproductive outcomes.
  6. Assisted Reproductive Technology (ART) Selection: Semen analysis plays a vital role in the selection of assisted reproductive technologies such as in vitro fertilization and gamete intrafallopian transfer technique, contributing to informed decision-making in fertility treatments.

Collection if Semen for Investigation of Infertility

Box 3: Semen analysis for initial investigation of infertility
  • Volume
  • pH
  • Microscopic examination for (i) percentage of motile spermatozoa, (ii) sperm count, and (iii) sperm morphology

A semen specimen is procured following approximately 3 days of sexual abstinence, as this duration optimizes the motility of sperm. Prolonged periods of abstinence can compromise sperm motility, while shorter intervals yield lower sperm counts. The collection process involves masturbation, utilizing a clean, dry, sterile, and leakproof wide-mouthed plastic container. It is imperative to transport the sample to the laboratory within 1 hour of collection.

The entirety of the ejaculate is gathered, with emphasis on the first portion due to its concentrated nature and the highest sperm count. During transit to the laboratory, it is crucial to maintain the specimen as close to body temperature as possible, achieved, for instance, by carrying it in an inside pocket. Ideally, specimen collection should occur near the testing site, preferably in an adjoining room.

Condom collection is discouraged, as it contains spermicidal agents that can impact the integrity of the sample. Ejaculation following coitus interruptus is not advisable, as it results in the loss of the initial concentrated portion of the ejaculate. Therefore, this method should be avoided for specimen collection.

To ensure accuracy and reliability, two semen specimens should be examined, collected 2-3 weeks apart. If there are significant variations in results, additional samples are warranted for a comprehensive assessment.

Examination of Seminal Fluid

Box 2 outlines the various tests applicable to seminal fluid, with a focus on those pertinent to infertility showcased in Box 3. The standard analysis involves assessing semen volume, sperm count, sperm motility, and sperm morphology.

For a comprehensive understanding of the terminology utilized in semen analysis, refer to Box 4. This terminology serves as a crucial reference in the nuanced assessment of seminal fluid, especially in the context of fertility-related investigations.

Examination of Semen to Check the Effectiveness of Vasectomy

Box 4: Terminology in semen analysis
  • Normozoospermia: All semen parameters normal
  • Oligozoospermia: Sperm concentration <20 million/ml (mild to moderate: 5-20 million/ml; severe: <5 million/ml)
  • Azoospermia: Absence of sperms in seminal fluid
  • Aspermia: Absence of ejaculate
  • Asthenozoospermia: Reduced sperm motility; <50% of sperms showing class (a) and class (b) type of motility OR <25% sperms showing class (a) type of motility.
  • Teratozoospermia: Spermatozoa with reduced proportion of normal morphology (or increased proportion of abnormal forms)
  • Leukocytospermia: >1 million white blood cells/ml of semen
  • Oligoasthenoteratozoospermia: All sperm variables are abnormal
  • Necrozoospermia: All sperms are non-motile or non-viable

The objective of post-vasectomy semen analysis is to ascertain the presence or absence of spermatozoa. Standard follow-up protocols involve initiating semen analysis approximately 12 weeks after the vasectomy procedure, or after about 15 ejaculations have occurred. If two consecutive semen samples yield negative results for sperm, the semen is deemed devoid of sperm.

Some recommendations advocate a follow-up semen examination at the 6-month mark to proactively rule out the possibility of spontaneous reconnection. This additional step in the follow-up process contributes to a more thorough and comprehensive assessment of the vasectomy's efficacy.

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