Chapter 14: Identification of Semen

14.1: Biological Characteristics

• A typical ejaculation releases 2–5 mL of semen, which contains seminal fluid and sperm cells.

• A normal sperm count ranges from 107 to 108 spermatozoa per milliliter of semen.

• Spermatogenesis: The process of generating spermatozoa.

• Seminal fluid is a complex mixture of glandular secretions.

• Prostatic fluid secretions account for approximately 30% of the ejaculate.

  • This portion of semen contains high concentrations of acid phosphatase (AP) and prostate-specific antigen (PSA).

• Vasectomy: The surgical removal of a bilateral segment of the ductus deferens. It prevents spermatozoa from reaching the distal portions of the male reproductive tract.

• Oligospermia: The condition by which males have abnormally low sperm counts.

• Azoospermia: A condition that causes males to produce no spermatozoa.

  

  

Spermatozoa

• The head contains a nucleus with densely packed chromosomes.

• Acrosomal Cap: A membranous compartment containing enzymes essential for fertilization found at the tip of the head.

• The head is attached to the middle piece through a short neck where the mitochondria that provide the energy for moving the tail are located.

• The tail or flagellum is responsible for spermatozoon motility.

  

Acid Phosphatase

• Acid phosphatase (AP) consists of a group of phosphatases with optimal activity in an acidic pH environment.
• The greatest forensic importance of AP is that prostate-derived AP contributes most of the AP activity present in semen.
• The half-life of AP activity at 37°C is 6 months.
• Elevated levels of prostatic AP found in serum are useful in diagnosing and monitoring prostate carcinoma.

Prostate-Specific Antigen (PSA)

• It is a major protein present in seminal fluid at concentrations of 0.5–2.0 mg/mL.
• PSA is produced in the prostate epithelium and secreted into the semen. It can also be found in the paraurethral glands, perianal glands, apocrine sweat glands, and mammary glands.
• PSA is a protein that has a molecular weight of 30 kDa and is thus also known as P30. It is responsible for hydrolyzing semenogelin (Sg), which mediates gel formation in semen.
• It is a member of the tissue kallikrein family and is encoded by the KLK3 locus located on chromosome 19.

Seminal Vesicle–Specific Antigen

• Human seminal vesicle–specific antigen (SVSA) includes two major types, semenogelin I (SgI) and semenogelin II (SgII).
• In humans, both SgI and SgII are present in a number of tissues of the male reproductive system, including the seminal vesicles, ductus deferens, prostate, and epididymis.
• The concentration of Sg in seminal fluid is much higher than that of PSA, and this is beneficial for the sensitivity of detection.
• Sg is present in seminal fluid and absent in urine, milk, and sweat, where PSA can be found.
• Although Sg compounds are present in skeletal muscle, kidney, and colon, this is not a great concern because these tissue samples are not routinely collected for semen detection in sexual assault cases.

14.2: Analytical Techniques for Identifying Semen

Presumptive Assays

• Lighting Techniques for Visual Examination of Semen Stains

  • A dried semen stain fluoresces under certain light sources such as ALSs or argon lasers.
  • ALSs are most commonly utilized for the visual examination of semen stains,
  • Excitation wavelengths between 450 and 495 nm can be used, allowing for the visualization of fluorescence with orange goggles.
  • The intensity of the fluorescence can be affected by different colors of substrates, and the material, such as clothing, where semen stains have been deposited.

• Acid Phosphatase Techniques

  • Colorimetric Assays

  • The AP contained in semen can hydrolyze a variety of phosphate esters.

  • It catalyzes the removal of the phosphate group from a substrate.

  • An insoluble colored precipitate at sites of acid phosphatase activity is formed with a stabilized diazonium salt.

    

  • Fluorometric Assays

  • These are more sensitive than the colorimetric detection of AP and are used for semen stain mapping.

  • AP catalyzes the removal of the phosphate residue on a 4-methylumbelliferone phosphate (MUP) substrate a reaction that generates fluorescence under ultraviolet light.

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Confirmatory Assays

• Microscopic Examination of Spermatozoa:

  • The cells from a questioned stain on an absorbent material can be transferred to a microscope slide by extracting a small portion of a stain with water, followed by gentle vortexing.
  • The suspension is then transferred to a slide and evaporated at room temperature or fixed with low heat.
  • The most common staining technique is the Christmas tree stain.
  • Nuclear Fast Red (NFR): A dye used for staining the nuclei of spermatozoa in the presence of aluminum ions.
  • Picroindigocarmine (PIC): A green component that stains the neck and tail portions of the sperm.
  • Epithelial cells appear blue-green and have red nuclei.
  • Fluorescent detection utilizing SPERM HY-LITER Fluorescent Staining Kit can facilitate the identification of spermatozoa.
  • Laser capture microdissection (LCM) has been shown to be an effective technique for separating spermatozoa from nonsperm cells.
  • This technique involves using a thin layer of a thermosensitive polymer that is placed on the surface of an LCM cap.

• Identification of Prostate-Specific Antigen

  • Immunochromatographic Assays:
  • PSA-Check-1, Seratec®
  • PSA Semiquant
  • One Step ABAcard PSA®

• Identification of Seminal Vesicle–Specific Antigen

  • Immunochromatographic Assays: RSID®-Semen test and Nanotrap Sg.

  • ELISA: Antibody Sandwich ELISA

  • The intensity of the signal can be detected and is proportional to the amount of bound antigen. The amount of PSA can also be quantified by comparing a standard with known concentrations.

    

    

• RNA-Based Assays

  • The essays are based on the expression of certain genes in a certain cell or tissue types.
  • The techniques used in the identification of semen are based on the detection of specific types of mRNA expressed exclusively in spermatozoa and in certain cells of male accessory glands.

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Application of RT-PCR for Semen Identification

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