Semen Analysis Workshop – Module 2

Objectives of Module 2

Master microscopic evaluation and documentation of seminal fluid, specifically:
- Sperm concentration determination
- Motility counting and calculation
- Modal progression assessment (progressive vs. non-progressive)
- Total motile count (TMC) computation
- Detailed sperm morphology scoring
- Viability (vitality) testing principles
- Proper semen centrifugation and pellet search when no sperm are initially seen

General Preparation & Quality Control

Always start with a thoroughly mixed specimen to ensure homogeneity.
Inspect immediately for agglutination or aggregation; note findings.
Use only clean, dry counting chambers and slides.
Record abstinence period and elapsed time between ejaculation and analysis; both influence reference‐limit interpretation.

Counting Chambers & Loading Technique

Common chambers:
- Makler (10 µm depth, 0.01 mm² squares)
- Leja disposable slides (20 µm depth variants)
- 10 µm or 20 µm deep Neubauer‐style slides
Standard loading for Makler:
- Pipette exactly 5 µL onto the central circular platform; allow coverslip to spread drop evenly.
- Avoid bubbles or overflow.
Counting protocol (Makler):
- Count motile and non-motile sperm in one full row (10 squares).
- Repeat on a second, different row (total 20 squares).

Sperm Concentration Calculations

Raw count → concentration because the Makler grid factor is 1 × 10⁶/mL per sperm seen per square.
Example slide shows final concentration reported as 6.6\ \text{million mL}^{-1}.

Percent Difference Between Two Counts

Formula:
(\%\;\text{diff}) = \frac{2(a-b)}{a+b} \times 100
where a = larger count, b = smaller.
Worked example (from slide):

a = 80.8,\ b = 74.5
a-b = 6.3
\frac{a+b}{2} = 77.7
\frac{6.3}{77.7} = 0.081
0.081 \times 100 = 8.1\% difference (acceptable reproducibility <10%).

WHO 5th Edition Reference Limits (selected)

Volume ≥ 1.5\ \text{mL}
pH ≥ 7.2
Concentration ≥ 15\ \text{million mL}^{-1}
Progressive + non-progressive motility ≥ 40 %; progressive alone ≥ 32 %
Vitality (live) ≥ 58 %
Morphology (strict) ≥ 4 % normal forms
WBC < 1\ \times 10^{6}\;\text{mL}^{-1} (peroxidase-positive cells)

Motility & Modal Progression

WHO advocates two categories: progressive (PR) vs. non-progressive (NP) + immotile (IM).
Erin White’s workshop scale (matches older WHO):
- 2 = definite forward progression (PR)
- 1 = weak/sluggish forward progression (NP)
- 0 = no forward progression (IM)
Example report: 50 % motile (PR + NP) with modal progression rating 2 and ≥32 % progressive fraction.

Total Motile Count (TMC)

Formula:
{\text{TMC}} = \text{Volume (mL)} \times \text{Concentration (million mL}^{-1}) \times \text{Fraction Motile}
Report table expresses TMC in \times 10^{8} per ejaculate.

Morphology (Strict / Tygerberg Criteria)

Normal form requirements:
- Oval head: length 5–6 µm, width 2.5–3.5 µm.
- Acrosome: 40–70 % of head area.
- ≤1–2 vacuoles, each ≤1 µm; larger or multiple = abnormal.
- Smooth, symmetric contour; no regional flattening.
- Midpiece length ≈ 6–10 µm (often cited 6–7 µm); aligns with head axis.
- Flagellum length 45–50 µm, uniform thickness, no kinks/coils.
Any deviation in head, midpiece, tail, or excess residual cytoplasm (>1/3 head size) categorizes sperm as abnormal.
Perfect heads with tail defects should be listed separately under morphology comments.

Slide Preparation

Two options:
1. Use commercially pre-stained morphology slides (rapid, consistent).
2. Manually smear sample, air-dry, then apply a Papanicolaou-type stain (Diff-Quik, Sperm Stain, etc.).
Minimum 200 sperm should be scored to reach 95 % confidence in % normal figure.

Abnormal Forms (illustrated in slides)

Head: tapered, pyriform, round, amorphous, small/no acrosome, vacuolated.
Neck/midpiece: bent neck, asymmetrical insertion, thick or thin midpiece.
Tail: short, bent, coiled, duplicated.
Excess residual cytoplasm droplet (>1/3 head).

Viability (Vitality) Testing

Definition: sperm with intact membranes (alive) regardless of motility status.
Immotile sperm cannot be assumed dead; must confirm with a vital stain or hypo-osmotic swelling (HOS) test.
Common dye: eosin-nigrosin (live sperm exclude eosin, remain white; dead sperm take up dye, appear pink/red).
IVF/ICSI Limitation: dyes generally forbidden during clinical fertilization work because residual stain may harm embryos.

Centrifugation & Pellet Search

If no sperm seen in counting chamber:
- Centrifuge entire specimen (e.g., 3000 g for 15 min).
- Discard supernatant, resuspend pellet in small volume (≈0.1 mL).
- Scan pellet thoroughly at high magnification to detect rare sperm (cryptozoospermia).

Practical / Clinical Interpretation Tips

High viscosity (rating 1) hampers accurate motility and concentration measurement; enzymatic or mechanical treatment may be required.
Percent difference between duplicate counts >10 % signals inadequate mixing or counting error; repeat.
Low concentration (<15 million mL⁻¹) and low TMC (<20 million/ejaculate) correlate with reduced natural fertility; informs physician about need for ART.
Morphology <4 % normal significantly lowers fertilization probability but must be interpreted with motility and concentration (complete picture).
Presence of WBC ≥1 × 10⁶ mL⁻¹ suggests leukocytospermia; consider cultures or antibiotics.

Ethical & Laboratory Considerations

Accurate, reproducible counts underpin physician counseling; errors may lead to unnecessary anxiety or treatment delays.
Documentation should mimic physician communication style—clear numerical results plus concise interpretive comments.
Vital staining helps lab decision-making yet must not contaminate gametes destined for ART.

Key Numerical & Formula Recap

Makler factor: 1 cell/square = 1\times 10^{6}\;\text{cells mL}^{-1}
Percent difference formula: \frac{2(a-b)}{a+b}\times100
TMC: \text{Volume}\times\text{Concentration}\times\text{Fraction Motile}
WHO lower reference limits: Volume 1.5 mL, Conc. 15 M/mL, Motility 40 %, Progressive 32 %, Morphology 4 %, Vitality 58 %.