Spermatogenesis

Question 1

Relevance of pH of sperm

Answer 1

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.

Question 2

Sperm parameters WHO 2021

Answer 2

volume = 1.4ml
Total sperm number = 39million per ejaculate
Sperm concentration = 16million/ml
Total motility = 42%
Progressive motility = 30%
Normal forms (morphology) = 4%

Question 3

Relevance of fructose in sperm

Answer 3

Low or absent fructose of indicates obstructive azoospermia (ejaculatory duct obstruction, CBAVD, retrograde ejaculation.

Question 4

Anatomy of male genital tract

Answer 4

Question 5

Components of macroscopic sperm evaluation

Answer 5

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.

Question 6

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

Answer 6

1. No spermatozoa in the fresh ejaculate even after centrifugation and microscopic examination of the centrifuged pellet.
2. Concentration of spermatozoa <16million/mL
3. Percentage of progressively motile spermatozoa below the reference limit (<30% 2021 WHO). Absolute asthenozoospermia is the absence of motile spermatozoa in the ejaculate.
4. Percentage of morphologically normal spermatozoa is below reference limit of 4%
5. Concentration and percentage of progressive motile both below reference limit as above.
6. Concentration and percentage of normal morphology of spermatozoa both below reference limit as above.
7. Concentration, percentage of progressive motile and percentage of normal morphology all below reference limit as above.
8. Low percentage of live and high percentage of immotile spermatazoa in the ejaculate
9. Concentration, motility and morphology all within normal limits.
10. Presence of spermatozoa in the ejaculate with small acrosome vesicles or total absence of the acrosome vesicle.
11. Presence of leucocytes in the ejaculate above the threshold of 1million/mL
12. No spermatozoa in the fresh ejaculate but spermatozoa are observed after microscopic examination of centrigued pellet.

Question 7

Assessment of sperm motility

Answer 7

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.

Question 8

Assessment of sperm vitality

Answer 8

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

Question 9

Sperm number and concentration

Answer 9

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.

Question 10

Morphology

Answer 10

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

Question 11

Indices for assessment of morphological abnormalities in sperm

Answer 11

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.

Question 12

azoospermia

Answer 12

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

Question 13

leuococytes/pyosperms

Answer 13

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.

Question 14

haematospermia

Answer 14

Often benign and self limiting
Causes:
infection
stones
trauma
obstruction - ejaculatory duct or urethral stricture
tumour
vascular
iatrogenic
uncontrolled hypertension

Question 15

Anti-sperm antibodies

Answer 15

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.

Question 16

Testing for ASA

Answer 16

Can be direct (only work with motile spermatozoa) or indirect

Direct – include MAR (mixed antiglobulin reaction) and the IB test (immunobead). MAR is performed on a fresh sample. IB on a washed sample.
MAR test is commercially available, inexpensive, quick and sensitive.

In the MAR test, a “bridging” antibody (anti-IgG or anti-IgA) is used to bring the
antibody-coated beads into contact with unwashed spermatozoa in semen bearing
surface IgG or IgA. The direct IgG and IgA MAR tests are performed by mixing
fresh, untreated semen separately with latex particles (beads) or treated red blood
cells coated with human IgG or IgA. A monospecific anti-human-IgG or anti-humanIgA is added to the suspensions. The formation of mixed agglutinates between
particles and motile spermatozoa indicates the presence of IgG or IgA antibodies
on the spermatozoa.

The direct immunobead test
This assay is more time-consuming than the MAR test but provides information about antibodies on spermatozoa that have been removed from possible masking components in seminal plasma. In the direct IB test, beads coated with covalently bound rabbit anti-human immunoglobulins against IgG or IgA are mixed directly with washed spermatozoa. The binding of beads with anti-human IgG or IgA to motile spermatozoa indicates the presence of IgG or IgA antibodies on the surface of the spermatozoa.

Indirect testing
The indirect IB test is used to detect ASAB in heat-inactivated, sperm-free fluids (serum, testicular fluid, seminal plasma or bromelain-solubilized cervical mucus). In this assay, sperm-free body fluid is incubated with washed donor sperm to allow ASAB present to bind to the sperm. Antibody-free donor’s spermatozoa take up ASAB present in the tested fluid and are then assessed as in the direct IB test.

Question 17

Blood testis barrier

Answer 17

Physical barrier between blood vessels and the semniferous tubules.
Walls of the seminiferous tubules are lined by germ cells and Sertoli cells. Barrier is formed by tight junctions, adheren junctions and gap junctions between the sertoli cells.
It divides the semniferous tubule into a basal compartment (outside of the tubule in contact with blood and lymph) and an endoluminal compartment.

Question 18

Function of the BTB

Answer 18

Allows Sertoli cells to control the adluminal environment in which germ cells develop by influencing the chemical composition of the luminal fluid.
The barrier also prevents passage of cytotoxic agents in.
Fluid in the lumen very different from plasma - very little protein and glucose but is rich in androgens, oestrogens, K+, inositol, gutamic and aspartic acid. This is maintained by the BTB
Protects against blood borne noxious agents
Prevents antigenic products of germ cell maturation from creating an autoimmune response,
May help establish an osmotic gradient that facilitates movement of fluid into the tubular lumen