Comprehensive Notes on Semen Analysis 35

Fitz's Investigations

  • The aim is to analyze Fitz's investigations to determine the cause of a couple's difficulty in conceiving, and whether assisted pregnancy techniques would be benificial.

Infertility due to Medical Treatments

  • Chemotherapy and radiotherapy for malignancy can cause infertility.
  • Cryopreservation of sperm before treatment allows for future IVF (in vitro fertilization) options.
  • Confirm no viable sperm is being produced.

Semen Analysis Volume

  • Volume less than 1.5 mL with a low sperm count may indicate:
  • Testosterone deficiency.
  • Duct blockage.
  • Problem with vas deferens development.

Volume and Sperm Count Expectations

  • Successful fertility or pregnancy not needed for investigation.
  • Expected volume: greater than 2 mL.
  • Sperm count: greater than 20 million (20 \times 10^6).
  • Count between 5 and 20 million: conception may take longer.
  • Count less than 5 million: natural conception is unlikely.

Motility

  • Motility should be greater than 40%.
  • Motility between 20% and 40%: conception may take longer.
  • Motility less than 20%: natural conception is problematic.

Semen Analysis Timing

  • Some tests must be done immediately to avoid lab-induced abnormalities.
  • Other tests can be done later.

Initial Volume Measurement

  • Measure volume by weight within the first 5 minutes.
  • Pre-weigh container, then weigh again with sample.
  • Subtract container weight to calculate volume.

Subsequent Assessments (30-60 minutes)

  • Assess liquefaction.
  • Examine macroscopic appearance.
  • Prepare a wet preparation.
  • Assess microscopic appearance and motility.
  • Determine necessary dilution for sperm count.

Additional Tests

  • Perform vitality test if indicated.
  • Assess sperm concentration using various dilutions if motility is low.
  • Prepare slides for morphology assessment.
  • Investigate for anti-sperm antibodies if immune response is suspected.

Reference Guide

  • Information from the Hope guide for examining and processing human semen.

Extended Analysis (Within 3 Hours)

  • Determine sperm concentration.
  • Send samples to micro lab if infection is suspected.

Later Analysis (Same Day)

  • Stain and assess morphology smears.
  • Perform biochemical analysis as needed.

Wet Preparation Procedure

  • Mix initial ejaculate sample and take aliquots.
  • Pre-warm sample, coverslips, etc. (temperature is important).
  • Initial examination under phase contrast at 100x magnification (10x objective, 10x optic).
  • Check for adequate mixing, distribution, and liquefaction.
  • Record any viscosity or clumping issues.

Higher Magnification Examination

  • Look for the presence of other cells.
  • Determine dilution for sperm count.
  • Assess motility.

Other Cells Present

  • Round cells: mostly immature germ cells (sperm progenitors).
  • Leukocytes (white blood cells): high levels may indicate infection or immune infertility.
  • Phagocytes: eliminate degenerating cells to maintain sperm quality.

Leukocytes and Sperm Quality

  • Leukocytes produce reactive oxygen species, impacting sperm quality and function.
  • Reactive oxygen species regulate capacitation (essential for fertilization).
  • Lower white cell levels may negatively affect IVF fertilization capacity.

Post-Vasectomy Samples

  • White blood cell count in semen decreases by up to 90%.
  • Indicates most white cells in semen originate from the testes.

Lifestyle Factors and Leukocyte Presence

  • Linked to leukocyte presence in semen.
  • Bacterial or viral infections.
  • Recovery from illness.
  • Increased venous issues around the testes (varicose veins).
  • Toxic therapy, cryptorchidism, maturation arrest.
  • Recreational drug use (alcohol, smoking, marijuana).
  • Trauma to the testes.
  • Frequent ejaculation.

Microscopic Examination of Sperm

  • Wet prep is good for assessing motility.
  • Morphology and concentration can be hard to assess due to movement.

Sperm Dilution Estimation

  • Estimate dilution from single high magnification field.
  • Greater than 200 sperm: use a 1 in 50 dilution.
  • Lower numbers: use a less dilute preparation.

Sample Preparation for Counting

  • Use at least 50 microliters of well-mixed ejaculate.
  • Total suspension examined should be at least 200 microliters.
  • Dilution immobilizes sperm and makes solution more aqueous for hemocytometer.
  • Prepare duplicate dilutions for analysis on both sides of counting chamber.

Sperm Clumping

  • Assess types of clumping separately.
  • Aggregation: sperm aggregated with epithelial cells, debris, or non-specifically with each other.
  • Agglutination: motile sperm sticking to each other (head-to-head, head-to-tail, mixed).

Agglutination Grading

  • Isolated: up to 10 sperm involved.
  • Moderate: 10 to 50 sperm involved.
  • Large: over 50 sperm involved.
  • Gross: all sperm agglutinated.

Progressive Sperm Motility

  • Total number of progressively motile sperm is biologically significant.
  • Calculated by multiplying total sperm count by percentage of progressively motile cells.
  • Temperature-dependent examination; warm sample slides and coverslips to 37 degrees Celsius.
  • Use a microscope with a graticule in the eyepiece to standardize the area.

Examination Procedure

  • Scan initially at 100x or 200x to check sperm density.
  • Move systematically, selecting fields at least 5 mm from cover slip edge.
  • Calculate the proportion/percentage of different categories; average replicates within an acceptable range.

Sperm Classification

  • Rapidly progressive: actively moving in a straight line or large circle, covering at least 25 microns (half their tail length) in a second.
  • Slowly progressive: moving in a straight line or large circle, traveling 5 to 25 microns in a second.
  • Non-progressive: moving in small circles or twitching tails, moving less than 5 microns in a second.
  • Immotile: no active tail movements.

Causes of Low Sperm Motility

  • Problems with Mitochondrial function (altered mitochondrial function).
  • Viscosity characteristics of seminal fluid (if the seminal fluid was too viscous it would be hard for the sperm to travel).
  • Antibody presence.
  • Infection or inflammation in the seminal tract.
  • Genetic factors (affecting mitochondrial DNA, ion channels or flagella proteins)

Genetic Factors Affecting Motility

  • Gene mutations in ion channels (proteins).
  • Gene mutations in flagella proteins.
  • Sperm proteins for cilia are common in other ciliated cells; motility problems could be linked to ciliated dyskinesia.
  • Large deletions or point mutations in genes encoding proteins in the oxidative phosphorylation pathway (cytochrome c oxidase or ATP synthase genes).

Sperm Vitality Assessment

  • Determined by assessing membrane integrity using a vital dye (dye exclusion test)
  • Perform sperm vitality test if less than 40% of cells are motile
  • Mix equal volume of semen with stain, incubate for a few seconds, make a smear on glass slide
  • Examine at 1000x magnification (100x oil immersion lens) under bright field
  • Count stained (dead) and unstained (alive) cells.

Sperm Count and Concentration

  • Affect timed pregnancy and pregnancy rates.
  • Correlates with testicular volume and testes capacity to make sperm.
  • Concentration is influenced by the volume of secretions.

Hemocytometer Counting

  • A central area containing 25 squares, each with 16 smaller squares.
  • Only count whole sperm (head and tail).
  • Record "pinhead sperm" if many heads without tails are seen
  • Report sperm heads with more than one tail if they are more than 20% of sperm.
  • Look at upper left area, determine how many sperm are present, which tells how many cells to look at.

Number of Sperm to Count on Hemocytometer

  • Less than 10 sperm: Count the whole grid.
  • Between 10 and 40: Count ten of the larger squares.
  • Greater than 40 sperm: Count five squares (four corners and center).
  • If less than 200 sperm are counted in total, extend your count to the next number.

Hemocytometer Sperm Counting Rules

  • Count all sperm within the central black line including any on the middle white line.
  • Don't count any sperm that are on the left-hand side/lower line
    *Follow the same procedure as blood cell counting to improve representative outcome.

Morphology Assessment

  • Morphology is assessed under light microscopy, using air-dried stained preparations.
  • The recommended stain is Papanicolaou. Quick differential stains may be used as well.
  • With Papanicolaou staining, the head appears blue and the mid-piece red; the tail may be blue or red. Excess cytoplasm will be green if normal and red if abnormal.
  • Slides can be stored with clear nail polish. Examined at 100x using bright field and oil immersion lens.
  • Look at at least 200 intact, mature sperm that are not overlapping. Report heads without tails only if they comprise over 20% of the total.
  • Morphological assessment may be of limited prognostic value for general pregnancy, but it can contribute. Normal level is around 3 to 5% morphologically normal sperm for sucessful pregnancy.

Sperm Morphology

  • Even if the morphology is normal, the sperm might have motility problems or DNA damage
  • The term "teratozoospermia" describes having high number of sperm that look abnormally shaped, linked to fertility. Can be caused by injuries, toxins, medicine, heat exposure, hormonal imbalances, lifestyle, oxidative stress, and infection/inflammation.

Normal Sperm Head Expectations

  • Regularly Contoured and Smooth.
  • Oval Pattern.
  • 40 to 70% of Head Should be Acrosome Region.
  • Up to Two vacoules.
  • Vacoules should not be more than Fifth of Sperm Head.

Microcephalic Definition

  • Defined as a sperm that is less than 3.5 microns.
  • Width is usually less than 2.5 microns.

Defects and Associated Genes

  • Rounded, Globozooid Sperm Heads: The sperm are usually deficient, without an acrosome. The syndrome is very rare impacting 0.1% of infertile men. Spata, FAT16, AND DPY19L2 are all genes associated with this syndrome.
  • Very Large Irregular Heads: These sperm heads are typically associated with macrocephalic sperm syndrome. AURKC is associated with mutations in the aurora kinase C gene.
  • Decapitated With No Head: Pinhead sperm are sperm with defecitive spermatogenesis . Identified Genes that have been associated are: Hook One Gene, and PUMFBP1 Gene.

Neck and Mid-Piece Abnormalities

  • Normally, the mid-piece should be slender, regular-shaped, and the same length as the sperm head. It should align straight with the head.
  • Abnormalities include irregularly shaped, too thin/thick, asymmetrical, abnormally angled.

Tail Abnormalities

  • Normally, the tail should be the same thickness down the whole length. Should be slightly thinner than mid-piece/ 45 microns, and ten times the head length. It can be looped, but not a very severe and sharp angle, as this can inhibit movement.
  • Severe angles of the tail can indicate problems/ dysfunction, such as coilness or dysplasia.

Cytoplasm

  • The acceptable amount is less than a third of size of sperm head, and anything above this is considered defective and may be associated with defective spermatogenesis .
    Reactive oxygen species also occur during this process which is triggered by cytoplasmic droplets .

Analyzing Sperm

  • Normal Sperm, Abnormalities Of Head, Tail, In Between Neck and Midpiece or Cytoplasm. This is recorded through multi-key cat which records/ tallies the different abnormalities. This process is done over a light microscope.
    *It is ensured this process is only counted once and is done over the process with recording what area is abnormal. To have the most accuracy during this you should be counting at least 200.

Sperm Analysis Flowchart

  • The given cell is assessed whether it can be classified. If it can't, this would be ignored; if classification is possible, then you check light prism to see large amount is residule.If this is the case, then it's scored. If this checks out, the same method/ analysis is done over individual parts of the sperm to see whether its tail, midpiece, or any other defects.
  • This systematic approach has it's individual drawbacks, as it cannot be determined with certainty, if this approach is objective. The assessment is also a lack in disagreement between individual instutions, or individual lab, as well as different staining that is provided.

Computer-Assisted Sperm Analysis (CASA) and Teratozoospermia Index (TZI)

  • CASA is designed to reduce operator variability and improve results and has now improved overall. In 2023, Tetra, also known as Fermi Index, was recently added with WHO guidelines.
  • WHO has not defined normal values for semen parameters. Reference limits are determined and those test centers below are defined as not definitively cut off as that can reduce sperm with impaired fertility. Severe defects, give much more evidence that the reasom for male infertilitly has much more evidence.