SEMEN ANALYSIS Flashcards
INDICATIONS
to check for infertility
to evaluate success of post vasectomy procedure
for Medico-legal cases/forensic studies
site of spermatogenesis; produces 5% of semen volume (sperm cells)
Testis (seminiferous tubules)
site of sperm maturation and storage
Epididymis
provide nutrients(fructose) for sperm
Seminal Vesicles
coagulation and liquefaction
Prostate gland
produce acidic fluid that contains ACP, citric acid, zinc, proteolytic enzymes
Prostate gland
add alkaline mucus to neutralize prostatic acid and vaginal acidity
Bulbourethral glands/ Cowper’s land
produce testosterone
Leydig cells
provide nutrients for the germ cells as they undergo spermatogenesis
Sertoli cells
propel sperm to the ejaculatory duct
Vas deferens / ductus deferens
COMPOSITION
Spermatozoa/Semen:
Seminal fluid/Seminal vesicles:
Prostate fluid.Prostate gland:
Alkaline mucus:
Epididymis, vas deferens, bulbourethral glands:
Spermatozoa/Semen: 5%
Seminal fluid/Seminal vesicles: 60-70%
Prostate fluid.Prostate gland: 20-30%
Alkaline mucus: 5%
Epididymis, vas deferens, bulbourethral glands: 10-15%
- produced by the seminiferous tubules of the testes
Sperm cells
CHEMICAL CONSTITUENTS
Acid phosphatase
Zinc
Fructose
Potassium, citric acid, ascorbic acid
Proteolytic enzymes
Spermine and choline
distinguishes semen from other fluids
Acid phosphatase
major nutrient of spermatozoa
Fructose
liquefaction and coagulation of seminal fluid
Proteolytic enzymes
Abstinence period:
3 to 5 days or not more than 7 days
Abstinence period Prolonged:
higher volume, decreased sperm motility and increased flavin content
Time of collection:
preferably early in the morning with an empty bladder before ejaculation
Container:
wide-mouthed, warm, sterile glass or plastic
Transport and testing: brought and tested in the lab within
30 minutes to 1 hour
Specimens for fructose level analysis should be tested within
2 hours
Fertility testing: requires [?] at 2 weeks interval
2 to 3 samples
is essential
Complete collection of semen
First portion not collected (1st and 2nd fractions)
Falsely decreased sperm count
Falsely increased pH
Specimen will not liquefy
Last portion not collected (3rd fraction)
Falsely decreased semen volume
Falsely increased sperm count
Falsely decreased pH
Specimen will not clot
METHODS OF COLLECTION
Self-Production or Masturbation
Condom Collection
Vaginal Vault Aspiration
Coitus Interruptus
: best method collection because it prevents contamination
Self-Production or Masturbation
: requires the use of condoms that are non-lubricant-containing and made from polyurethane.
Condom Collection
: contains spermicidal agents
***Ordinary condoms
: aspiration of seminal fluid from the vaginal vault after coitus
Vaginal Vault Aspiration
: not reliable method of collection because the first portion of the ejaculate may be lost
Coitus Interruptus
All semen specimen are potential reservoirs for
HIV and HEPATITIS virus
STANDARD PRECAUTIONS
Discarded as
biohazardous waste
Specimens must be kept at
body temperature (37oC)
: preserved in frozen state at -85C and stored for one year
Artificial insemination
: should be frozen if delay in testing is unavoidable
Fructose level testing
PHYSICAL EXAMINATION
Performed after liquefaction (within [?])
30 to 60 minutes
Color
Grayish white to Pearly white
Transparency
Translucent
Volume
2-5mL/ejaculation
Volume
Measured using a
graduated cylinder with 0.1 increments
Volume
Increased:
Decreased:
Increased: prolonged abstinence
Decreased: infertility
Odor
Fishy, distinct, “Chlorox-like”, Musty, acrid
Viscosity
Highly viscous, pours in droplets
Viscosity
Normal:
viscid to highly viscid
Viscosity
Reporting:
0 (watery) – 4 (gel-like)
Prolonged [?] indicates a possible deficiency in prostatic enzymes
Liquefaction
Assessed by pouring semen into a graduated glass (normal: drop by drop)
Liquefaction
Incomplete [?] impedes sperm motility and should be reported
Liquefaction
Liquefaction Time
30 minutes-1 hour
pH
7.2-8.0
: possible infection within the reproductive tract
↑pH (alkaline)
: increased prostatic fluid , ejaculatory duct obstruction, poorly developed seminal vesicles
↓pH (acidic)
Specific Gravity
1.027 to 1.032
may indicate bleeding
Rust to red-brown
may indicate urine contamination, antibiotics, prolonged abstinence or pyospermia
Yellowish
may indicated infection and presence of WBCs
Increased white turbidity
infertility
Clear
MICROSCOPIC
Sperm motility
Sperm count
Sperm concentration
Sperm morphology
Sperm viability
Main factor which affects the penetrating ability of the sperm cells through the cervical mucus barrier
SPERM MOTILITY
Performed on a well-mixed liquefied specimen within 1 hour from time of collection, under 20 HPF
SPERM MOTILITY
SPERM MOTILITY
A [?] should be used
positive displacement pipette
SPERM MOTILITY
Coverslip:
22x22 mm
Evaluate the movement and direction
SPERM MOTILITY
SPERM MOTILITY
Alternative procedure: Examine [?] and count the percentages of the different motile categories using a manual cell counter
200 sperm per slide
SPERM MOTILITY
Normal: A minimum motility of [?] with a rating of [?] within [?]
50%
2.0
1 hour
WHO: within 1 hour, [?] or more of the sperm should be motile in categories a, b, and c, or [?] or more should show categories a and b.
50%
25%
Number of cells per ejaculate
SPERM COUNT
SPERM COUNT
Normal Value:
> 40 million/ejaculate
Number of cells per ml
SPERM CONCENTRATION
SPERM CONCENTRATION
Normal Value:
> 20 million/ml
NORMAL VALUES
Routine:
Kruger’s Strict Criteria:
> 30% normal
14% normal
involves measuring the head, neck, tail and acrosome
Kruger’s Strict Criteria
Diluting Fluids:
1.5% sodium bicarbonate
2.1% formalin
0.5% chlorazene
4.1% formalin in sodium bicarbonate
5% sodium bicarbonate + 1% phenol in distilled H2O
Chilled distilled H2O
Only [?] should be counted
fully developed sperm cells
[?] must not be included
Immature sperm and WBCs (“round cells”)
[?] can be counted if clinically significant
Spermatids (immature sperm cells) and WBCs
– inflammation or infection of the reproductive system that can lead to infertility
> 1 million WBCs per ml
– disruption of spermatogenesis
> 1 million spermatids per ml
Procedure: 1 drop of liquefied semen → pre-warmed slide → cover with a coverslip ringed with petrolatum
Hanging Drop Method
Count at least 200 spermatozoa under HPF
Hanging Drop Method
Motile: moving actively forward
Non-motile: sluggish in direction
Hanging Drop Method
Performed in an undiluted semen under approx 20 HPF
Routine Grading of Motility
Utilizes undiluted specimen
Makler
Sperm cells are immobilized by heating
Makler
Sperm cells are counted using the 4 corner squares and the large central square
Neubauer Hemocytometer
Both sides loaded
Neubauer Hemocytometer
Counts should agree within 10%
Neubauer Hemocytometer
Phase-contrast or bright-field microscopy
Neubauer Hemocytometer
- Decrease in the number of sperm cells or presence of few motile cells
Oligospermia
- seen in unilateral or bilateral hypotrophic testes and hypothyroidism
Oligospermia
- Complete or total absence of spermatozoa
Azospermia
- Seen in bilateral underdeveloped testes, obstruction and infection with gonorrhea
Azospermia
- Presence of dead or immobile sperm cells
Necrospermia
- Seen in hypertrophic testes and decrease in fructose levels
Necrospermia
- Oval shaped; measuring 5um in length and 3um in width
Head
- Acrosomal cap: should occupy 1/2 of the head and 2/3 of the nucleus
Head
- Attaches the tail to the head
Neck/Middle Piece
- Contains mitochondria that provides energy for flagellar motion
Neck/Middle Piece
- Approximately 45um in length and exhibits a whip-like motion that propels the sperm cell
Tail
Stain and evaluate at least
200 sperm cells under oil immersion field
Percentage of [?] is reported
abnormal sperm
Stains:
• Papanicolaou – stain of choice
• Giemsa stain
• Wright’s stain
Head:
double heads, giant and amorphous heads, pinheads, tapered heads and constricted heads
Tail:
doubled, coiled or bent
Neckpiece:
abnormally long
Provides objective testing sperm motility based on sperm velocity and trajectory (direction of motion)
Computer-Assisted Semen Analysis
Also determines sperm concentration and morphology
Computer-Assisted Semen Analysis
Differentiation and enumeration of round cells—
immature sperm cells(spermatids) and leukocytes
Variables:
• C = number of round cells
• N = number of spermatids and/or leukocytes counted
• S = sperm concentration in millions per ml
C = (N x S) / 100
Suspected if a specimen has normal sperm concentration with markedly decreased motility
I. Sperm Viability Test/Bloom’s test/ Eosin Negrosin
Assessed within 1 hour of ejaculation by mixing the specimen with Eosin-Nigrosin
I. Sperm Viability Test/Bloom’s test/ Eosin Negrosin
number of dead cells in 100 sperm is counted using a bright-field or phase contrast microscope
I. Sperm Viability Test/Bloom’s test/ Eosin Negrosin
I. Sperm Viability Test/Bloom’s test/ Eosin Negrosin
LIVING CELLS:
DEAD CELLS:
Normal:
bluish white in color
red against a purple background
75% living cells/100 sperm cells
Low levels are associated with androgen deficiency, low testosterone level and seminal vesicle insufficiency
Fructose Test
Samples tested within 2 hours or frozen to prevent fructolysis
Fructose Test
Resorcinol Test: 1 ml semen + 9 ml reagent –> boil –> orange red color
Fructose Test
Spectrophotometric: used to quantitate fructose level in semen
Fructose Test
Normal: ≥ 13 umol/ejaculate
Fructose Test
Destroys blood-testes barrier
Antisperm Antibodies (ASA)
Can be detected in men and women
Antisperm Antibodies (ASA)
screening procedure to detect IgG antibodies
Mixed agglutination reaction (MAR)
The semen sample is incubated with AHG and a suspension of latex particles or treated RBCs coated with IgG
Mixed agglutination reaction (MAR)
(+): >50% agglutination
Mixed agglutination reaction (MAR)
more specific test and detects IgG, IgM and IgA antibodies and demonstrate
Immunobead test
what area of the sperm the autoantibodies are affecting
(+): ≥ 20% sperm with bound immunobead
Immunobead test
Post-coital test to determine the quality of the cervical mucus and the ability of sperm cells to penetrate it
Sims-Huhner Test
Determines the presence of choline
Florence Test
Florence Test Reagents:
potassium iodide and iodine crystals
Florence Test (+) Result:
presence of brown rhombic or needle-like crystals under the microscope
Determines the presence of spermine
Barbiero’s Test
Barbiero’s Test Reagents:
picric acid and trichloroacetic acid
Barbiero’s Test (+) Result:
presence of yellow leaf-like structures
Determines the tenacity of the mucus
Spinbarkeit Test
Specimens are tested at monthly intervals, beginning at 2 months after vasectomy and continuing until two consecutive monthly specimens shows no spermatozoa
Post-vasectomy Semen Analysis