SEMEN ANALYSIS 2

Sperm Count and Concentration, Sperm Morphology

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 to 160/250 million/ml or > 20 million/ml

Diluting Fluids for Sperm Count and Concentration

(1) 1.5% sodium bicarbonate

(2) 2.1% formalin

(3) 0.5% chlorazene

(4) 4.1% formalin in sodium bicarbonate

(5) 5% sodium bicarbonate + 1% phenol in distilled H2O

(6) chilled distilled H2O

Sperm Count and Concentration Recommended Dilution:

1:20

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

decreased in the number of sperm cells or presence of few motile cells

Oligospermia

Oligospermia is seen in unilateral or bilateral

hypotrophic testes

hypothyroidism

Complete or total absence of spermatozoa

Azoospermia

Azoospermia is seen in

bilateral underdeveloped testes,

obstruction

infection with gonorrhea

Presence of dead or immobile sperm cells

Necrospermia

Necrospermia is seen in

hypertrophic testes and decreased in fructose levels

Oval shaped measuring 5um in length and 3um in width

Head

Acrosomal cap: should occupy ___ of the head and ___ of the nucleus

1/2 , 2/3

Located at the tip of the head and contains oocyte-penetrating enzymes necessary for ovum penetration

Acrosomal cap

Attaches the tail to the head

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

Stains for morphology examination

(1) Papanicolaou

stain of choice

(2) Giemsa stain

(3) Wright’s stain

Routine Criteria for morphological examination

>30% normal

Kruger’s Strict Criteria:

>14% normal

involves measuring the head, neck, tail and acrosome

Kruger’s Strict Criteria:

immature sperm cells(spermatids) and leukocytes

round cells

Suspected if a specimen has a normal sperm concentration with markedly decreased motility

Sperm Viability

Procedure: semen sample + Eosin-Nigrosin stain ———> smear preparation

Sperm Viability

LIVING CELLS stains

bluish white in color

DEAD CELLS stains

red against a purple background

Sperm Viability Normal value

75% living cells/100 sperm cells

Low levels associated with androgen deficiency, low testosterone level and seminal vesicle insufficiency

Fructose Test

Fructose Test methods

Resorcinol Test:

Spectrophotometric:

1 ml semen + 9 ml reagent ——→ orange red color

Resorcinol Test:

used as a screening test for fructose

Resorcinol Test:

used to quantitate fructose level in semen

Spectrophotometric:

Spectrophotometric Normal value:

≥ 13 umol/ejaculate

≥ 20 mU/ejaculate

Neutral α-glucosidase

≥ 2.4 umol/ejaculate

Zinc

≥ 52 umol/ejaculate

Citric acid

≥ 100 U/ejaculate

Acid phosphatase

Production of antisperm antibodies involves the disruption of the

blood-testis barrier

ASA Positive result

(+) clumps of sperm (agglutination) during routine examination

Detects surface IgG autoantibodies based on a modification of the Coombs test

Mixed Agglutination Reaction Test (MAR)

Involves both in vivo sensitization of sperm cells by ASA and in vitro sensitization of RBCs/latex beads

Mixed Agglutination Reaction Test (MAR)

Mixed Agglutination Reaction Test (MAR) (+) Result:

> 50% Agglutination

Proportions of spermatozoa coated with antibody

Immunobead Test

Immunobead Test Determines the antibody class:

IgG, IgA or IgM

Localization on the spermatozoa Head-directed Ab:

interferes with penetration

Localization on the spermatozoa Tail-directed:

affect motility through the cervical mucosa

Immunobead Test (+) Result:

≥ 20% sperm with bound immunobead

Immunobead Test Clinically Significant Result:

≥ 50% sperm with bound immunobead

Performed in specialized andrology laboratories for assisted reproduction

Sperm Function Tests

List Sperm Function Tests

Hamster Egg Penetration

Cervical Mucus Penetration

Hypo-osmotic Swelling

In-vitro Acrosome Reaction

Sperm cells are incubated with species-nonspecific hamster eggs and penetration is observed microscopically

Hamster Egg Penetration

Observation of sperm penetration ability of partner’s midcycle cervical mucus

Cervical Mucus Penetration

Sperm exposed to low-sodium concentrations are evaluated for membrane integrity and sperm viability

Hypo-osmotic Swelling

Evaluation of the acrosome to produce enzymes essential for ovum penetration

In-vitro Acrosome Reaction

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