Spermatogenesis Flashcards
Relevance of pH of sperm
pH of sperm depends on the relative contributions of prostatic and seminal vesicular fluid. Prostatic = acidic. Seminal vesicle = alkaline.
pH <7.2 = lack of alkaline seminal vesicular fluid (under-developed or absent) or their is urine contamination.
pH <7.0 with low volume and azoospermia = CBAVD.
pH should be measured within 30mins of SA collection.
Sperm parameters WHO 2021
volume = 1.4ml
Total sperm number = 39million per ejaculate
Sperm concentration = 16million/ml
Total motility = 42%
Progressive motility = 30%
Normal forms (morphology) = 4%
Relevance of fructose in sperm
Low or absent fructose of indicates obstructive azoospermia (ejaculatory duct obstruction, CBAVD, retrograde ejaculation.
Anatomy of male genital tract
Components of macroscopic sperm evaluation
Appearance – Usually homogenous, cream/grey opalescent appearance.
Abnormalities could include –
Less opaque (sperm concentration is very low)
Colour change –yellow after long abstinence
Red-brown – if RBC present
Clearer yellow – vitamins/drugs or patient jaundiced
Viscous/clear and colourless – pre-ejaculate only ? has the patient ejaculated for this sample
Liquefaction – should liquefy within 15-30minutes, if not this should be recorded.
Viscosity – Estimated by gently aspirating it into a wide-bore pipette, allowing the semen to drop by gravity and observing the length of any thread. If abnormal the drop will form a thread more than 2cm long.
Odour – if there is a strong odour of urine or putrefaction this should be recorded.
Terminology:
1. Azoospermia
2. Oligozoospermia
3. Asthenozoospermia
4. Teratozoospermia
5. Oligoasthenozoospermia
6. Oligoteratozoospermia
7. Oligoasthenoteratozoospermia (OAT)
8. Necrozoospermia
9. Normozoospermia
10. Globozoospermia
11. Leukocytospermia
12. Cryptozoospermia
- No spermatozoa in the fresh ejaculate even after centrifugation and microscopic examination of the centrifuged pellet.
- Concentration of spermatozoa <15million/mL
- Percentage of progressively motile spermatozoa below the reference limit (<30% 2021 WHO). Absolute asthenozoospermia is the absence of motile spermatozoa in the ejaculate.
- Percentage of morphologically normal spermatozoa is below reference limit of 4%
- Concentration and percentage of progressive motile both below reference limit as above.
- Concentration and percentage of normal morphology of spermatozoa both below reference limit as above.
- Concentration, percentage of progressive motile and percentage of normal morphology all below reference limit as above.
- Low percentage of live and high percentage of immotile spermatazoa in the ejaculate
- Concentration, motility and morphology all within normal limits.
- Presence of spermatozoa in the ejaculate with small acrosome vesicles or total absence of the acrosome vesicle.
- Presence of leucocytes in the ejaculate above the threshold of 1million/mL
- No spermatozoa in the fresh ejaculate but spermatozoa are observed after microscopic examination of centrigued pellet.
Assessment of sperm motility
Should be assessed as soon as liquefaction has occurred.
Velocity of motile spermatozoa is temperature dependent – therefore essential to standardize the temperature (similar to body temp)
5 fields evaluated. Count systematically all the rapidly and slowly progressive spermatozoa.
Four category grading system:
Rapidly progressive (>25um/s), spermatozoa move actively, either linearly or in a large circle, covering a distance, from the starting point to the end point, of at least 25um (1/2 tail length) in one second.
Slowly progressive (5-25um/s), spermatozoa moving actively, either linearly or in a large circle, covering a distance from the starting point to the end point of at least 5-25um (one head to less than ½ tail length) in one second.
Non progressive (<5 um/s) all other patterns of active tail movements with the absence of progression, i.e. swimming in circles, the flagellar force displacing the head less than 5um (one head length) from the starting point to the end point.
Immotile – no tail movements.
Assessment of sperm vitality
Important if <40% motile sperm as it distinguishes immotile dead sperm from immotile live sperm.
Assessed by identifying those with an intact cell membrane (eosin-nigrosin test)
Measure as soon as liquefaction occurs.
If >25-50% are alive but immotile - suggests primary ciliary dyskinesia
Sperm number and concentration
Meausred within 3 hours of specimen collection
Requires dultion with a fixative to immobilise the sperm to count accurately
Needs to be loaded into a haemocytometer - count at least 200 per replicate
Count only whole spermatozoa
Concentraion - spermatozoa per ml and total spermatozoa per ejaculate should be counted.
Morphology
1, Head - smooth contour, oval, acrosomal region 40-70% of head area, no vacuoles
2, Midpiece - slender, regular, same length as sperm head, major axis aligns with sperm head
3, Tail - uniform calibre, thinner than midpiece, 20x head length, can be looped but not sharply (broken flagellum)
4, Cytoplasmic residue - droplets <1/3rd sperm head size are normal
Indices for assessment of morphological abnormalities in sperm
Teratozoospermia index (TZI) – max 4 defects per abnormal spermatozoon (head, midpiece, principal piece and one for excess residual cytoplasm)
Multiple anomalies index (MAI) – similar to TZI based on mean number of anomalies per abnormal spermatozoon, all head, midpiece and principal piece anomalies are included.
Sperm deformity index (SDI) – total number of defects divided by the total number of spermatozoa.
azoospermia
can only use this term if no spermatozoa seen in the sediment of a centrifugesd sample.
1ml aliquot - centrifugation at 3000g for 15mins
NB centrifugation often results in loss of motility an underestimation of concetration
leuococytes/pyosperms
Not possible to distinguish between leucocytes and immature germ cells with routine smear - need speciliased peroxidase staining.
Should send for urine culture, urine PCR for chlamydia and gonorrhoea and semen culture.
Infection of male accessory glands is a reversible cause of subfertility.
data lacking about empirical treatment if no cause is found.
haematospermia
Often benign and self limiting
Causes:
infection
stones
trauma
obstruction - ejaculatory duct or urethral stricture
tumour
vascular
iatrogenic
uncontrolled hypertension
Anti-sperm antibodies
Caused by disruption in blood-testis barrier (vasectomy, trauma, biopsy, orchitis, torsion, cancer) - sperm antigens prime immune system
Found in only 2-4% of infertile couples
Can be found and quantified in serum, seminal fluid or directly bound to sperm.
Block penetration of cervical mucus, prevent fertilisation through interference with zona binding.
Measure in men with unexplainf infertility and men with isolated low motility.
No clear percentage threshold used to diagnose.
ASA bound to head is more significant than bound to tail. ASA to tail tip is not associated with infertility.
IgA and IgG - IgA clinically more important
Agglutination may be a sign of ASA but not all agglutinated samples have ASA and vice versa.
