Ch10 semenanalysis

Copyright © 2014. F.A. Davis Company
CHAPTER 10CHAPTER 10
SEMENSEMEN

Upon completing this chapter, the reader will be able to
1.State the structures involved in sperm production and their function.
2.Describe the four components of semen with regard to source and
function.
3.Explain the procedures for collecting and handling semen specimens.
4.Describe the normal appearance of semen and three abnormalities in
appearance.
5.State two possible causes of low semen volume.
6.Discuss the significance of semen liquefaction and viscosity.
Learning ObjectivesLearning Objectives

7. Calculate a sperm concentration and count when provided with the
number of sperm counted, the dilution, the area of the counting
chamber used, and the ejaculate volume.
8. Define round cells, and explain their significance.
9. State the two parameters considered when evaluating sperm motility.
10. Describe the appearance of normal sperm, including structures and
their functions.
11. Differentiate between routine and strict criteria for evaluating sperm
morphology.
12. Given an abnormal result in a routine semen analysis, determine
additional tests that might be performed.
Learning ObjectivesLearning Objectives (cont’d)(cont’d)

13. Describe the two methods routinely used to detect antisperm
antibodies.
14. List two methods for identifying a questionable fluid as
semen.
15. State the World Health Organization (WHO) reference values
for routine and follow-up semen analysis.
16. Discuss the types and significance of sperm function tests.
17. Describe methods of quality control appropriate for semen
analysis.
Learning ObjectivesLearning Objectives (cont’d)(cont’d)

• Assisted reproductive technology (ART)
• Abnormal results on the routine semen analysis
– Specialized andrology laboratories
• Fertility testing
• In vitro fertilization (IVF)
AndrologyAndrology

• Semen consists of four components
– Contributed separately by the
• Testes and epididymis
• Seminal vessels
• Prostate
• Bulbourethral glands
– Normal semen specimen must have all four
components
PhysiologyPhysiology

• Paired testes: located in scrotum
– Lower scrotum temperature optimal for sperm development
• Seminiferous tubules of testes
– Produce spermatozoa
• 5% volume
– Sertoli cells provide support and nutrients for the germ cells as they
undergo spermatogenesis
– Mature and stored in the epididymis
• Develop flagella
• Seminal vessels
– Produce majority of fluid (60% to 70%); transport medium
– Provide fructose and flavin for sperm metabolism
– Sperm are not motile without this fluid
PhysiologyPhysiology (cont’d)(cont’d)

• Prostate gland
– Produce acidic fluid (20% to 30%)
– Contains acid phosphatase, citric acid, zinc, and
proteolytic enzymes
– Enzymes coagulate semen prior to ejaculation and cause
liquefaction after ejaculation
• Bulbourethral glands
– Produce thick, alkaline fluid (5%) to neutralize acid from
prostate and acid pH of vagina
– Absence of fluid = diminished sperm motility
PhysiologyPhysiology (cont’d)(cont’d)

Structure Function
Seminiferous
tubules of testes
Spermatogenesis
Epididymis Sperm maturation
Ductus deferens Propel sperm to ejaculatory ducts
Seminal vesicles Provide nutrients for sperm and fluid
Prostate gland Provide enzymes and proteins for
coagulation and liquefaction
Bulbourethral
glands
Add alkaline mucus to neutralize
prostatic acid and vaginal acidity
Summary of Semen ProductionSummary of Semen Production

The Male GenitaliaThe Male Genitalia

• Must be a complete specimen
• First portion of the ejaculate is missing, then
– Sperm count will be decreased
– pH is falsely increased
– Specimen will not liquefy
• Last portion of ejaculate is missing, then
– Semen volume is decreased
– Sperm count is falsely increased
– pH is falsely decreased
– Specimen will not clot
• Detailed instructions to patients
Specimen CollectionSpecimen Collection

• 2 to 3 days abstinence; no longer than 7 days
– WHO recommends that two or three samples be collected not less
than 7 days or more than 3 weeks apart
• Laboratory provides containers and ideally a room for
collection
• Home: deliver to laboratory within 1 hour; keep specimen 37°C
• Record
– Patient name and date of birth
– Period of abstinence
– Time of collection and receipt
• Collected by masturbation
– If not possible, not lubricated, antispermicidal condoms
Specimen CollectionSpecimen Collection (cont’d)(cont’d)

• Standard precautions must be observed at all
times during analysis.
• Specimens are discarded as biohazardous waste.
• Sterile materials and techniques must be used.
Specimen HandlingSpecimen Handling

• Macroscopic and microscopic
• Appearance
• Volume
• Viscocity
• pH
• Sperm concentration and count
• Motility
• Morphology
Fertility EvaluationFertility Evaluation

• Normal is gray-white, translucent
• Musty odor
• White turbidity = infection
– Culture
– LE reagent strip
• Red = blood cells, abnormal
• Yellow = urine, prolonged abstinence, medications
• Urine is toxic to sperm: no motility
AppearanceAppearance

• Fresh semen specimen is clotted and should liquefy
within 30 to 60 minutes after collection
– Deficiency in prostatic enzymes
• Analysis cannot begin until liquefaction has occurred
• Enzymes added to induce liquefaction
– Document
– May affect biochemical tests, motility, and
morphology
LiquefactionLiquefaction

• Normal: 2 to 5 mL
• Measure in a graduated cylinder
• Increased volume may be seen following periods of
extended abstinence
• Decreased = infertility, incomplete collection
• Viscosity: droplets with thin threads from a pipette
are normal
– Form threads longer than 2 cm = highly viscous
• Rate 0 (watery) to 4 (gel-like) or low, normal, high
Volume and ViscosityVolume and Viscosity

• Measure within 1 hour of ejaculation
• Normal: 7.2 to 8.0
• Over 8.0 = infection
• Decreased pH = increased prostate fluid,
ejaculatory duct obstruction, or poorly
developed seminal vesicles
• Check with pH pad of a urinalysis reagent strip
pHpH

• Valid measurement of fertility
• Concentration = number sperm/mL
• Count = number sperm per ejaculate
• Total sperm count
– Sperm concentration × specimen volume
• Reference value: >20 to 250 million sperm per milliliter
– 10 to 20 million borderline
• Normal count = concentration × volume
– >40 million/ejaculate
Sperm Concentration and CountSperm Concentration and Count

• Concentration performed in Neubauer chamber
– Five small squares, corners, and center of large center
square
• Common dilution: 1 to 20
• Diluting fluid = sodium bicarbonate and formalin,
must immobilize sperm
• Count both sides of chamber, sides must agree
within 10%; use the average
• With this method, the number of sperm counted are
multiplied by 1,000,000 = sperm/mL
(cont’d)(cont’d)

Areas of the Neubauer Counting ChamberAreas of the Neubauer Counting Chamber
Used for Red and White Blood Cell CountsUsed for Red and White Blood Cell Counts

• Must multiply sperm per μL by 1000 to reach
sperm/mL
• Sperm count = sperm/mL × volume
– Example
- 60,000,000 sperm concentration × 3 mL volume =
180,000,000 sperm count/ejaculate
• Instrumentation has been developed; used
primarily in fertility clinics

• Need to have sperm with forward, progressive movement
• Well-mixed, liquefied semen specimen
• Examine within 1 hour; evaluate undiluted on glass slide with cover slip
• Estimate percentage with progressive, forward motion in 20 HP fields
• Or, examine 200 sperm per slide and count the percentages of the
different motile categories using a manual cell counter
• Grading can be done using a scale of 0 to 4, with 4 indicating rapid,
straight-line movement and 0 indicating no movement
• A minimum motility of 50% with a rating of 2.0 after 1 hour is
considered normal
Sperm MotilitySperm Motility

• World Health Organization (WHO)
recommendation
– Motility is graded as progressive motility (PM),
nonprogressive motility (NP), and immotility (IM);
motility must be specified as total motility (PM and
NP) or progressive motility (PM)
• Instrumentation available
– Computer-assisted semen analysis (CASA)
Sperm MotilitySperm Motility (cont’d)(cont’d)

• Critical to fertilization
• Evaluate head, neck, midpiece, tail
– Head: oval with acrosomal cap at end and covering half of the
head
• 5 µm long and 3 µm wide
• Contains enzymes for ovum penetration
– 7.0 µm long neckpiece attaches head to midpiece and tail
• Midpiece surrounded by sheath of mitochondria for tail movement
– Flagellar tail approximately 45 µm
– <<AU: should this slide be moved down with other Sperm
Morphology slides?>>
Sperm MorphologySperm Morphology

Spermatozoon StructureSpermatozoon Structure

• Observe on thin smear under oil immusion<<AU:
“immersion”?>>
• Wright’s, Giemsa, Shorr, or Papanicolaou stain
• Count 200 and report number of abnormal
• Routine criteria measures
– Abnormalities in head structure include double heads, giant
and amorphous heads, pinheads, tapered heads, and
constricted heads
– Abnormal sperm tails are frequently doubled, coiled, or bent
Sperm MorphologySperm Morphology

• Kruger’s strict criteria include
– Measuring head, neck, and tail size
– Measuring acrosome size
– Evaluating for the presence of vacuoles
• Strict criteria evaluation is an integral part of
assisted reproduction evaluations
• >30% normal forms when using routine criteria
• >14% normal forms when using strict criteria
Sperm MorphologySperm Morphology (cont’d)(cont’d)

• Round bodies
– White blood cells (WBCs) and spermatids (immature sperm)
– Differentiate on morphology smear
– Count number of each separately in 100 cells
• N is the number of spermatids or neutrophils counted per 100 mature
sperm,
• S is the sperm concentration in millions per milliliter
C = N × S
100
Normal: <1,000,000

Sperm VitalitySperm Vitality
• Eosin-nigrosin stain
• Dead cells stain red;
normal are blue-white
• Count number/100 cells
• Normal: 75% living
• Corresponds to motility
• Seminal fructose tests

• Low sperm concentration
• Low to absent fructose level in the semen
• Lack of the support medium
– Abnormalities of the seminal vesicles
– Bilateral congenital absence of the vas deferens
– Obstruction of the ejaculatory duct
– Partial retrograde ejaculation
– Androgen deficiency
Seminal Fluid FructoseSeminal Fluid Fructose

• Present in both men and women
• Male more common: surgery, vasectomy
reversal, trauma
• Sperm normally do not encounter the immune
system, so body considers them foreign
• Damaged sperm create female antibodies
• Suspect male antibodies when clumps of sperm
are seen; female no clumping
Antisperm AntibodiesAntisperm Antibodies

• Female: mix female serum with sperm and check for
agglutination
• Immunoassays: males
• Mixed agglutination reaction (MAR) test
– Incubate sperm with antihuman globulin (AHG) and IgG-
coated latex particles
– AHG combines with particles and antibody-coated sperm
forming clumps
• Normal: <10% motile sperm attach to particles
Antisperm AntibodiesAntisperm Antibodies (cont’d)(cont’d)

• Immunobead test
– Demonstrates antibodies to head, neck, midpiece,
and tail
– Beads are coated with antihuman globulin
– Microscopic examination shows where on sperm
antibodies are attacking
– Tail = movement; head = penetration
– Normal: beads on <20% of sperm
Antisperm AntibodiesAntisperm Antibodies (cont’d)(cont’d)

• >1 million WBCs usually indicate prostate
infection
– Culture and test for Mycoplasma hominis, Chlamydia
trachomatis, Ureaplasma urealyticum
• Chemistry tests: neutral α-glucosidase, zinc, citric
acid, acid phosphatase
– ↓ Neutral α -glucosidase = epididymis
– ↓ Zinc, citric acid, acid phosphatase =↓ prostatic
fluid
Microbial and Chemistry TestingMicrobial and Chemistry Testing

• Neutral a-glucosidase ≥20 mU/ejaculate
• Zinc ≥2.4 µmol/ejaculate
• Citric acid ≥52 µmol/ejaculate
• Acid phosphatase ≥200 units/ejaculate
Normal Semen Chemical ValuesNormal Semen Chemical Values

• Semen present in specimen?
• Observe microscopically for sperm
– Enhance with xylene; use phase microscopy
• Specimen acid phosphatase content
• Detection of seminal plasma glycoprotein, p30
• DNA analysis
Semen DetectionSemen Detection

• Only concern is presence of sperm
• Takes several months for all sperm to be gone,
based on time and ejaculations
• Begin in 2 months; continue until 2 months are
negative
• Wet preparation under phase; if negative,
centrifuge for 10 minutes, examine again
• Only one sperm is required for fertilization
Postvasectomy AnalysisPostvasectomy Analysis

• Clinical Laboratory Improvement Amendments
(CLIA) rates semen analysis as high complexity
• Commercial controls and training materials are
available
• CAP <<AU: Spell out CAP>> offers proficiency
testing
<<AU:defined CLIA; ok?>>
Semen Analysis Quality ControlSemen Analysis Quality Control

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