Semen Analysis

TERMS:

Azoospermia: absence of spermatozoa in semen.

aspermia: no semen

Spermatid: immature sperm cell

Necrospermia: the presence of dead sperm cells

Oligospermia: decreased number of sperm

Asthenozoospermia: low percentage of progressively motile spermatozoa

Teratozoospermia: low percentage of morphologically normal spermatozoa

Seminal fluid or semen

The composite solution formed by the testes as well as other male reproductive organs.
Consists basically of spermatozoa suspended in seminal plasma

Stages of Passage of Sperm Cells

Testis
- Male reproductive gland that produces sperm cells and secretes testosterone.
- Site of spermatogenesis in seminiferous tubules.
Epididymis
- Storage and maturation of sperm cells.
Vas Deferens
- Transports sperm from the epididymis to the ejaculatory duct.
Seminal Vesicle
- Provides fructose and other components to nourish sperm and forms part of semen.
Ejaculatory Duct
- Conducts sperm combined with seminal fluid toward the prostate gland.
Prostate Gland
- Adds slightly acidic fluid containing enzymes for sperm motility and viability, further contributing to semen.
Bulbourethral Gland (Cowper's Gland)
- Produces alkaline fluid to protect sperm and lubricate the urethra.
Urethra
- Final passage through which semen is expelled from the body during ejaculation.

IMPORTANCE:

Important for fertility studies
Follow-up a post-vasectomy procedure
- This evaluation typically occurs at monthly intervals beginning two months after the procedure, and a successful vasectomy is indicated by two consecutive monthly specimens showing no spermatozoa.
for forensic studies (eg. rape & paternity)
Causes of Infertility
Varicocele - most common

forms when valves inside the veins along the spermatic cord prevent blood from flowing properly.
Swelling and Widening of the veins (hardening of veins that drain the testes)
Tapering of the head of the sperm cells
1. Mumps
2. Klinefelter's syndrome
3. Malignancy

Sperm cell maturation

Spermatogenesis

- formation and development of sperm in the seminiferous tubules of the testes

Spermiogenesis

- Maturation of spermatids into sperm cells

NOTE: The entire process takes approximately 90 days

Sperm Cell Maturation Stages:

Spermatogonium: The initial germ cells located in the seminiferous tubules of the testes that undergo mitosis to produce more spermatogonia.
Primary Spermatocyte: Formed from spermatogonia, these cells undergo the first meiotic division to produce two secondary spermatocytes.
Secondary Spermatocyte: Resulting from the division of primary spermatocytes, these undergo the second meiotic division leading to the formation of spermatids.
Spermatid: The early stage of sperm cells; they are immature and have round shapes. They will undergo further maturation in the epididymis.
Sperm Cell: The final maturation stage where spermatids, through a process called spermiogenesis, develop their distinctive shape, develop a tail, and acquire motility, resulting in fully mature sperm cells.

Sertoli cells provide support and nutrients for the germ cells as they undergo mitosis and meiosis

Testis

Male reproductive gland
Produces sperm cells
Secretes testosterone
Site of spermatogenesis
(Seminiferous Tubules)
5% of the entire ejaculate
Epididymis
is a coiled tube located behind the testis.
Responsible for the storage and maturation of sperm cells.
It plays a crucial role in the male reproductive system by allowing sperm to undergo further development and gain motility prior to being transported through the vas deferens.
Seminal vesicle
Convoluted, pouch-like
Alkaline, viscous fluid
- transport medium for the sperm
Fructose: provides nutrients to the sperm cells
Prostaglandin: for sperm motility & viability
Clotting proteins: coagulation of semen after ejaculation
- After semen is expelled, it initially coagulates to form a gel-like structure, which is commonly referred to as a coagulum. This coagulum later liquefies to become serum-like, allowing the sperm to swim freely.
Flavin: gray or opalescent appearance of semen
- green-white fluorescence under UV light

Prostate gland

Golf ball
Milky
Slightly acidic fluid (6.5)
Citric acid - used by sperm for ATP production
Prostatic specific antigen
Proteolytic enzymes break down clotting proteins liquefaction of semen
Acid phosphatase
Zinc
Choline and spermine: inhibit bacterial growth
Bulbourethral gland or Cowper's gland
Pea -size
Alkaline fluid
protects the passing of sperm by neutralizing acid from urine in the urethra
Secretes mucus- lubricates the penis & the lining of the urethra

Collection

The collection should be complete
Entire specimen in a container
First portion: sperm cells and prostatic fluid

When part of the first portion of the ejaculate is missing, it can lead to several consequences in the analysis of the semen sample:

Sperm Count Decreased: The first portion of the ejaculate typically contains a higher concentration of sperm. If this portion is missing, the overall sperm count in the analysis will appear lower than it actually is, leading to a false impression of low fertility.
pH Falsely Increased: The seminal fluid composition includes prostatic fluid that contributes to the normal pH range. Missing the first portion may result in an insufficient amount of acidic prostatic fluid, leading to an artificially inflated pH reading, which could suggest a problem that isn't actually present.
Specimen Will Not Liquefy: The initial part of the ejaculate contains enzymes that are crucial for liquefaction. If this portion is missing, the semen may not liquefy properly, complicating the analysis and potentially masking underlying issues with the sample.

If part of the last portion of the ejaculate, which contains most of the fluid from the seminal vesicle, is missing, it can lead to several consequences for the semen analysis:

Decreased semen volume: This indicates that not all constituents of the semen are present, which can affect fertility assessments.
Falsely increased sperm count: The overall sperm concentration in the analysis may appear higher than it actually is due to the missing portion that usually contains a higher concentration of sperm.
Falsely decreased pH: The absence of seminal fluid that helps maintain the normal pH can result in inaccurately low readings, potentially misleading assessments related to infections or obstructions.
Specimen will not clot: The crucial enzymes for the coagulation process may be lacking, complicating the analysis and masking potential issues.
As indicated in the requisition form:
Patient's name and birth date
Period of sexual abstinence
Completeness of the sample
Difficulties with collection (eg. When pressured the specimen may contain RBCs)
Time of specimen collection and specimen receipt (Time is crucial, pag labas input agad ng time)

Collection Methods for Semen Sample:

Masturbation (Self-Production): This method involves the individual collecting the semen sample through self-stimulation.
After Interrupted Sexual Intercourse (Coitus Interruptus): Semen is collected after the male partner withdraws before ejaculation.
Condom: A collection condom must be free from spermicidal agents, as usual condoms often contain these chemicals. Only non-lubricant polymeric silicone (Silastic) or polyurethane condoms should be used.
Aspiration from the Vaginal Canal After Intercourse: Involves collecting semen from the vaginal canal after intercourse.

Requirements prior to collection:

Empty bladder before ejaculation
Sexual abstinence: 2 to 7 days
- Prolonged: higher volume; decreased motility
Fertility testing: For accurate assessments, two or three samples should be provided.
- Samples should not be less than 7 days or more than 3 weeks apart.
- Significant result: AT LEAST 2 abnormal samples
Warm sterile glass or plastic containers (plastic has an inhibitory effect on motility after 60 minutes)

Requirements for transport:

kept at room or body temperature 37°C.
Delivered to the laboratory within 1 hour of collection.
Specimen Handling:
Observe standard precautions.
Potential reservoir for HIV and hepatitis viruses.
Discarded as bio-hazardous waste.

Semen Analysis

Basic Examinations: WHO
Sperm concentration
Motility
Morphology Percentage

SEMEN ANALYSIS

Macroscopic Examination

1. Appearance/ Color

2. Volume

3. Liquefaction Time

4. Viscosity

5. pH

Microscopic

1. Motility

2. Morphology

3. Count

Appearance

Normal Gray white/pearly white translucent (with musty odour)
Turbid: indicates WBC’s; infection
Clear: low sperm count
Yellow: urine contamination; prolonged abstinence, medications
Gray: flavin

Liquefaction

Normal Timeframe: Expected to occur 30 to 60 minutes after sample collection.
Prolonged Liquefaction: If liquefaction takes longer, this may indicate a deficiency in prostatic enzymes.
Analyzing Specimens: Ensure to analyze the specimen only after it has liquefied.
NOTE: Action if Unliquefied After 2 Hours: If the specimen has not liquefied after two hours, consider adding Dulbecco's phosphate-buffered saline or proteolytic enzymes like alpha chymotrypsin bromelain to facilitate the process.

Volume

clean graduated cylinder

0.1-mL increments

Normal: 2 to 5 ml per ejaculate

Low volume: non-adherence to 2-day abstinence

- Infertility

- problem in the seminal vesicle

High volume: signifies prolonged abstinence

Viscosity

Pour the specimen, observe how it pours

Normal viscosity = pours like droplets

Highly viscous =droplets with threads >2 cm

Rating of 0 if watery to 4 if gel-like

Can also be reported as low, normal, or high viscosity

Highly clumped and viscous semen- slower sperm motility

Normal - 7.2 -8.0

High pH: infection

(reproductive tract)

Low pH: increased prostatic fluid

obstruction of the ejaculatory duct

problem in the seminal vesicle

Methods:

pH pad of urinalysis gt strip

pH paper

II. MICROSCOPIC EXAMINATION

Sperm Concentration/ Count

Concentration is per ml, while count is as the whole specimen

Reference value: RBC pipette is used

Sperm conc. 20 to 250 million sperm per mL

Borderline: 10 to 20 million per mL

Sperm count: at least 40 million/ejaculate

Common dilution 1:20

mechanical (positive-displacement) pipette

10uL semen

190 uL cold water

Formula:

Add specimen and cold water divided by the specimen used

Dilution = to immobilize the sperm prior to

counting

Diluting fluids

- sodium bicarbonate (dissolves mucus) and formalin (immobilizes sperm)

- chilled water

- saline

/ Neubauer chamber

/ Count only the mature cells not the immature

"round cells"

The middle of the three lines defines the square's boundary (black line, left panel). Al spermatozoa within the central square are counted, as well as those with their heads between the two inner lines (white circles), but not those whose heads lie between the outer two lines (black circies). A sper-matazoon with most of its head lying on the central line is counted only ll that line is the lower ar left-hand line of the square (white circles, middle panel) but not if it is the upper or right hand line of the square (black circles, right panel).

Sperm Concentration/ Count

Using 2 WBC squares

Sperm Conc./ml = # of cells counted X DF (20) X 1,000

# of squares(2) X vol of 1 square (0.1)

= # of cells counted X depth(10) X Dil (20) X 1.000

Sperm Conc./ml=# of sperm cells counted X 100,000

Sperm count/ejaculate sperm conc. X volume

Shortcut:

Sperm count x 100,000

RBC square

1:20 dilution

5 RBC square x 1 million

Makler Counting Chamber

for counting sperm cells uses a cover plate with 1mm grid divided into 100 squares

uses undiluted specimen

The number of spermatozoa counted in any strip of10 squares of the grid indicates their concentration in millions/mL.

No additional factors are necessary for calculation

Causes of Low sperm count

ENVIRONMENTAL CAUSES

Industrial chemicals Heavy metal exposure Radiation or X-rays

Overheating the testicies

-HEALTH, LIFESTYLE AND OTHER CAUSES

Drug use Anabolic steroids cocaine or manjuana

Alcohol use

Tobacco smoking Emotional stress

Weight

2. Sperm Motility

sperm must penetrate the cervical mucosa to the uterus, fallopian tubes and ovum

Hanging Drop Method

Undiluted, well mixed and fully-liquefied semen

10ul and 22 X 22 coverslip, concave slide

Stand for 1 minute

Observe in 20 high power fields

% of motile sperm cells

Normal > 50 % within 1 hr

Sperm

Motility Interpretation

Normal (within 1 hour)

50% or more: motile in categories a, b, and c

25% or more: show progressive motility (a and b)

Observe for immobile sperm and clumps (Sperm antibodies)

High percentage of immobile sperm?

Perform sperm vitality testing

Clumping of sperm?

Possible presence of sperm agglutinins

Alternative sperm grading criteria

Recommended by WHO

Motility must be specified as:

Total motility (PM and NP) or Progressive

PM)

Computer-assisted semen analysis (CASA) performed under 75 seconds

Sperm velocity and trajectory (direction of motion)

Sperm concentration

Sperm morpholology

3. Sperm Morphology

Observe for head, neckpiece, midpiece and tail under OlO

200 sperms are evaluated

Note the percentage of

normal and abnormal forms

Stains :

Papanicolaou

Giemsa

Hematoxylin

3 Distinct Parts

1. head - for ovum penetration

oval-shaped 5um long and 3um wide

acrosomal cap (40-70%) ½ of head

neckpiece

middle piece - 7 um contains mitochondria that provide energy for flagellar tail motion

3. tail - 40-45 um long, for motility

Head abnormalities - poor ovum penetration

Tail abnormalities- affect motility of the sperm

Kruger's-Strict criteria

- recommended by WHO

- not routinely performed

: a system of evaluating sperm morphology using morphometry and stage micrometer strictly measuring head, neck, tail, acrosomal head and vacuoles.

normal forms(strict criteria) 14%

normal forms(routine criteria) 30%

SQA-V Sperm Quality Analyzer

Automatic results in 75 seconds Sperm concentration

Motility (PM+NP)

Immotility (IM)

% normal morphology

Sperm motility index (SMI)

Average velocity (VELOCITY)

Total sperm concentration (TSC)

Motile sperm concentration

(MSC)

Progressively motile sperm concentration (PMSC)

Spermatid

round, condensed nucleus

more than 1 million spermatids/mL indicates disruption of spermatogenesis

Causes: viral infections exposure to toxic chemicals genetic disorders

Neutrophil

more than 1 million WBCs/mL

indicates an inflammatory condition associated with infection and poor sperm quality and may impair sperm motility and DNA integrity

Sperm Vitality (Viability)

Bloom's Test

Eosin/Nigrosin Test

Done in cases of infertility where the sperm count is normal but has decreased motility

living sperm cells: not infiltrated by eosin remain bluish white in color

dead sperm: red (stained by eosin)

Evaluate 100 sperm cells

Normal: 50% or more living cells

High number of vital but immobile cells defective flagellum (Motility problem)

High number of immotile and non-viable cells epididymal pathology (problem is epididymis)

Seminal Fluid Fructose

Done in cases where the sperm count is low

Evaluates capacity of seminal vesicle to provide fructose to sperm (Nutrition)

tested within 2 hours or frozen to prevent fructolysis

Resorcinol test: Orange (red-orange) color

Quantitative: spectrophotometric methods

Normal : ≥ 13 umol per ejaculate

Retrograde ejaculation:

the semen travels backwards into the bladder

Florence Test

- uses Florence rgt (lodine crystals & KI)

(+) brown rhombic crystal (periodide of choline)

Barbiero's test

- uses TCA & picric acid

(+) yellow leaf like crystals (spermine picrate)

Leukocytes

Leukocyte esterase reagent strip is used as screen for the presence of leukocytes

Anti-Sperm Antibodies (Clumping of sperm cells)

Blood testes barrier: should be intact

to prevent sensitizing the immune system from producing antibodies against own sperm

following surgery, vasectomy reversal (vasovasostomy), trauma and infection can compromise blood testes barrier

the antigens on the sperm produce an immune response that damages the sperm

1. Mixed agglutination reaction (MAR)

screening procedure to detect the presence of immunoglobulin G (IgG) antibodies

semen sample containing motile sperm incubated w/ IgG antihuman globulin (bivalent AHG) and latex particles or treated RBCs coated with IgG.

AHG binds simultaneously with both Abs on sperm and on latex particles.

(+) result - clumps of sperm & particles or cells

2. Immunobead Test

more specific,

detects IgG, IgM, and IgA Abs.

demonstrates the area of the sperm affected by antibodies

Head region - penetration into the cervical mucosa or ovum.

Tail region - movement of the sperm

uses Polyacrylamide beads coated with either anti-IgG, anti-IgM or anti-lgA.

result shows the beads attached to affected sperm's particular area

(beads on < 50% of sperm is normal)

reported as " IgM tail Abs" or " IgG head Ab" etc

WBC count

More than 1 million WBC IS SIGNIFICANT

Commonly diseased organ is the prostate

C. trachomatis, M. hominis, Ureaplasma urealyticum

Detection of semen in Medico Legal Cases

1. Microscopic Exam for presence of sperm cells

specimen with xylene and examining under

phase microscopy

Motile sperm - up to 24 hours

Non-motile sperm - 3 days

Heads of sperm remain - 7 days

2. prostatic acid phosphatase

3. seminal glycoprotein p30 (prostatic specific antigen [PSA])

4. ABO blood grouping & DNA analysis

Post-vasectomy Evaluation

Evaluates presence or absence of sperm

Monthly intervals beginning at 2 months postvasectomy

Successful vasectomy: 2 consecutive monthly specimens show no spermatozoa

Wet preparation, phase contrast

If negative, centrifuge for 10 min

then re-examine

Spinnbarkeit Test o test for the tenacity of cervical mucus

good: 10 cm before breaking

Sim Huhner Test ( Post Coital Test)

Measures the quality of cervical mucus and the ability of the sperm cells to penetrate the mucus and maintain activity

NV 6-8 hrs after coitus

10 motile sperm cells /hpf

Chemical testing

Disorder of the seminal vesicle

: decreased fructose

Disorder of epididymis:

decreased neutral a glucosidase. glycerophosphocholine and L carnitine

ck of prostatic fluid: decreased zinc, citric acid glutamyl trans peptidase acid phosphatase