Seminal Fluid Analysis

• Seminal fluid is another type of body fluid

Objectives

• The function of reproductive structures for seminal fluid formation can be described
• Composition of seminal fluid can be discussed
• Seminal fluid analysis can be described including collection, physical, chemical and microscopic evaluation
• Morphologic appearance of normal and abnormal forms of spermatozoa can be described and identified
• The origin and clinical significance of cells other than sperm in the seminal fluid can be discussed

Clinical Significance

• Reasons for seminal fluid analysis include fertility testing, and postvasectomy semen analysis
• Forensic analyses can be conducted including medicolegal cases, and alleged rape
• Seminal fluid analysis can be used for selection of donors for therapeutic insemination, in-vitro fertilization or embryo transfer
• It can be used for exposure screening to reproductive toxicants
• and for cryopreservation or sperm banking

Male Reproductive System

• Male reproductive system includes the ureter, urinary bladder, ductus deferens and seminal vesicle
• Additional components include prostate gland, bulbourethral gland, urethra and epididymis
• The testis, penis and glans penis are also components
• The sagittal section includes the pubic bone, and scrotum

Definition of Terms

• Seminiferous tubules are the site of spermatogenesis and spermatozoa production and regulated by Sertoli cells
• Sertoli cells play a role in supplying nutrients, hormones and other necessary substances for spermatogenesis or nurse cells for developing sperms
• The epididymis is where sperm maturation and storage occurs, and where sperm gain motility before ejaculation
• Interstitial cells of Leydig produce and secrete testosterone
• The vas/ ductus defens propels sperm to the ejaculatory duct
• Seminal vesicles makes up 60-70% of seminal fluid, provides nutrients such as fructose for sperm, alkaline fluid, fructose, prostaglandins and flavin
• Fructose provides energy for sperm
• Flavin imparts the characteristic gray or opalescent to light yellow, and green-white fluorescence under UV
• Prostaglandin lowers the female immune response to the semen.
• Phosphorous and potassium contributes to sperm motility
• Proteins play a role in coagulation of ejaculate, creating a gel-like protective layer around sperm
• The prostate gland contributes 20-30% to seminal fluid
• Prostatic fluid secretions aid coagulation and liquefaction
• Principal components of the milky, slightly acidic fluid: citric acid, enzymes (ACP and proteolytic enzymes), and proteins for coagulation
• ACP can be used to identify semen
• Proteolytic enzyme can be used for liquefaction
• Zinc is used to evaluate prostate function and a decreased level of zinc is associated with the prostate gland disorders
• Bulbourethral gland contributes 5% and adds alkaline mucus to neutralize prostatic acid and vaginal acidity
• Azoospermia describes the total absence of sperm cells that results from bilaterally small, underdeveloped testes, ejaculatory obstruction from traumatic or operative procedures or secondary to gonorrhea
• Oligospermia is characterized by presence of a few motile cells and low sperm count from bilateral or unilateral hypotrophic testes; and may be secondary to hypothyroidism
• Necrozoospermia describes a condition in which there is a low percentage of live and a high percentage of immotile or dead spermatozoa in semen
• Asthenozoospermia describes inability of vital sperm cells to move. (slow moving sperm cells or "lazy" sperm cells)

Factors Affecting Semen Quality

• The 1st portions of semen are prostatic secretions rich in sperm cells
• If a portion of the initial ejaculate is not collected, there is decreased volume, decreased sperm concentration, and increased pH
• If the semen coagulum will fails to liquefy
• Later fractions/last portion contains seminal vesicular fluid
• If the last portion of ejaculate is missing there is decreased semen volume, increased sperm contraction and a coagulation will not form
• Losing the first portion of the ejaculate has more influence on the results of semen analysis than losing the last portion
• Increased volume of seminal fluid enhances sperm motility = dilution may occur and underestimate sperm concentration
• Abstinence for a minimum of 2 days is needed
• Lacking at least 2 days of abstinence can affect sperm number and quality
• Abstinence for a maximum of 7 days is needed
• Prolonged abstinence increases volumes and reduces motility or increases flavin
• Absence of ejaculation causes spermatozoa to accumulate in the epididymis, overflow into the urethra and have tendency to be flushed into the urine
• Penultimate abstinence period can affect sperm count, as a single ejaculation will not clear all sperm cells
• As the epididymis are not completely emptied by ejaculation with some spermatozoa remain from the previous
• The size of the testis influences the total number of spermatozoa per ejaculate
• Size also reflects the level of sperm activity which also affects sperm, morphology
• Takiara demonstrated that the size of the testes bears a direct correlation with testicular function
• The product of masturbation correlates to a lower quality, in a room near laboratory; sperm
• Product of sexual activity corresponds to higher quality. Condoms during intercourse

Specimen Collection

• Instructions should be clearly written and spoken concerning the collection of the sample. Ensure the sample is complete and any loss of any fraction of the sample should be reported
• Abstinence for a minimum of 2 days, and a maximum of 7 days of sexual abstinence is needed
• The collection site should be a private room near the laboratory to limit the exposure of the semen to fluctuations in temperature and to control the time between collection and analysis
• Report form should document the name of the patient and date of birth, period of abstinence and date/time of collection
• Additional information should include completeness of sample (any producing difficulties), and the time interval between collection, and the start of semen analysis
• Complete collection is needed, the bladder should be empty before ejaculation, as urine is toxic to sperm
• 2-3 samples within a 3-month period and at least 7 days apart should be examined for fertility testing
• A clean wide mouthed container made of glass or plastic that has been confirmed to be non-toxic for spermatozoa should be used for collection
• A pre-weighed container can be labeled with the name and ID number of the subject
• Transport to and preserve the sample, in the laboratory within one hour of collection
• Keep sample at ambient temperature between 20 and 37 degrees celsius (close to body temp) while waiting for analysis
• Artificial insemination requires samples to be preserved and frozen at -85 degrees Celsius
• The best collection method is masturbation, or a nonlubricated rubber or polyurethane condom
• Ordinary latex condoms are not acceptable because they interfere with the motility of spermatozoa
• Coitus interruptus: not a reliable method as the first portion of ejaculation contains highest number of spermatozoa
• The low pH of vaginal fluid affects sperm motility
• Vaginal vault aspiration: Seminal fluid from the vaginal vault after coitus
• Semen samples may contain infectious agents such as HIV, HBV, and herpes simplex virus
• Handle specimens as biohazards and discard as waste
• Use sterile materials and techniques for bioassay, intra-uterine insemination, in-vitro fertilization or intracytoplasmic sperm injection

Physical Examination

• Semen analysis should begin within 30 to 60 minutes right after liquefaction
• Liquefaction happens immediately after ejaculation forming a semi-solid coagulated mass
• At room temperature, semen usually begins to liquefy (become thinner), with a heterogeneous mixture of lumps in the fluid
• Normal samples may contain jellylike granules, but mucus strands interfere with semen analysis
• Failure of liquefaction can be caused by deficiency in prostatic enzymes and should be reported
• Adding an equal volume of Dulbecco’s phosphate -buffered saline, followed by pipetting can aid in liquefaction
• Reducing inhomogeneity: Repeated gentle passage through gauge 18/19 attached to syringes
• Viscosity
  • Normal viscosity is defined by small discrete drops
  • Abnormal viscousity is defined by thread >2 cm long causing interference with sperm motility/concentration
  • viscosity is reported using values 0 or 4
    • 0: Watery
    • 4: Gel-like
  • Alternate reporting: normal is slightly viscous, moderately viscous or unable to aspirate into pipette
• Color and Appearance
  • Homogeneous, grey-opalescent appearance is normal
  • Normal
    • Turbid = WBCs (infection)
    • Rusty red/red-brown = RBCs (hemospermia)/drugs like pyridium
    • Yellowish = Flavin (prolonged abstinence) + urine contamination, jaundice, multivitamins
    • Clear = infertility
• Volume:
  • 1.5 - 5 mL is normal
  • Low (hypospermia)
    • Infertility, Ejaculatory duct obstruction, absence of vas deferens, loss during collection, androgen deficiency
    • Active inflammation, prolonged abstinence is high
  • Volume is measured by weighing pre-weighed container
    • OR collecting sample into tube/cylinder
  • Volume in mL can be derived: Weight in mL/ Density in g/mL. Semen density=1g/mL ranges 1.043-1.102 g/mL
• Odor
  • Odor should be distinct musty/bleach-like
  • Abnormal means something infectious

Chemical Examination

• pH
  • pH ranges between 7.2 - 8.0
  • Infections cause elevated pH in reproductive tract (bacteria=alkaline)
  • Decreased pH comes from ejaculatory duct obstruction OR increased secretions

Microscopic Examination

• phase contrast microscope is recommended for all examinations of unstained preparations of fresh semen at a total magnification of x400 (at x1000 if stained preparation)
• Microscopic examination of semen involves sperm motility, vitality, concentration and morphology
• Inspected at a total magnification of x100; LPO
• Provides an overview of the sample, especially the number of spermatozoa, the presence of mucus strands, sperm agglutination or aggregation
• Determines cells other than spermatozoa (epithelial and round cells) leukocyes/germ cells
• Wet prep: Semen sample mixed well and the components are distributed
  • It is mixed 10 times by aspirating with disposable pipette
• aliquot of semen (10 ul) is added after mixing
  • transferred to a clean side with coverslip
• fixed depth of 20 mm to allow spermatoza to swim freely
  • rotating movement is constrained at <20 mm
  • Aggregation is the adherence of either immotile/motile spermatozoa to other
  • Agglutination: Refers to motile spermatozoa sticking

Sperm Motility

• Graded traditionally: Examining in an undiluted specimen, determining percentage and quality
• Graded using computer-spermed analysis(CASA)
• Evaluate immediately for liquefication of sample-preferably within 30m/1hr. Standardization
• Performed at rm temp or 37 deg- may need to standardize based on each lab
  • Rapid progression, some lateral movement
• B is slower, some lateral movement
• C is noticeable lateral and slow
• Progressive motility: Spermatozoa move in a line/large circle in a steady fashion
• Non-progressive motility: sperm exhibits movement by either small circles and force barely displaces head/observation of flagella

Sperm Concentration

• Locate grid to check there should be 5 squares
• Dilutions need to be carried out based on the average # if Spermatoza
• average less than 101= 1:20(50 mL in 950ul fixative)
  • Normal values limits (WHO). Number over 15 x 10^6/mL.
  • Total value over 39 x 10^6/ejaculate Calculations
  • Sperm concentration (per ml) = Total # rows (R1+R2)/Total number of cells X VCFxDF
  • Df- dilution factors= total of solution / volume. VCF( volume correction factor)- Vol wanted/ volume used Sperm #- Sperm concentration x semen vol

Sperm Morphology

• Kruger's strict # is 14%. Routine criteria # >= 4%. Can have defects to tails, neck, cytoplasm.