Diagnostic Assessment

Question 1

What is the first diagnostic step in male fertility investigation?

Answer 1

-analysis of seminal fluid and sperm parameters as an indicator of male fertility potential using WHO criteria for normal semen parameters (2021)

Question 2

What are different types of semen analysis?

Answer 2

1. manual semen analysis (preferred in clinical setting)
2. computer- assisted semen analysis (CASA)

Question 3

How is manual semen analysis carried out?

Answer 3

• lab practitioner uses light microscope for cell count
  -used more inc clinical setting

Question 4

How does CASA work?

Answer 4

• you click a button to eject a slide from the box below the monitor, add your slide, close it, then click the same button , you can use knobs to alter focus.
  used more in research setting

Question 5

WHO reference values (2021)

Answer 5

Volume - 1.5-6.0ml
appearance/colour - grey opalescent appearance
liquefaction - <30 minutes
sperm conc - >/= 15 million/ml
motility >/ 40%
morphology >/- 4%
Vitality - > 54%
pH - 7.2 - 8
leucocytes - <1 million/ml

Question 6

Assessment of appearance

Answer 6

• grey- opalescent appearance

Question 7

what are 2 methods of measuring sperm volume?

Answer 7

1. Direct volume measurement method :
   - volume (ml) measured directly using a serological pipette
2. volume from weight:
   - weighing pots before and after sample production
   -difference = sample volume
   -studies have shown weight to be acurate indication of volume, 1g = 1ml

Question 8

Assessment of liquefaction

Answer 8

-normal liquefaction should take place within 20-30 minutes post production (abnormally long liquefaction time may be indicative of an infection eg. bacterial prostatis)

Question 9

What are 2 devices used to measure sperm concentration?

Answer 9

1. Neubauer haemocytometer - the one we use, grids and look at dilution factor.
2. Makler counting chamber

Question 10

Assessment of concentration

Answer 10

sperm conc = quantity of sperm present in a sample
- measured in millions per ml
- determined using counting chamber

Question 11

What are samples below the reference value concentration known as?

Answer 11

-oligozoospermia (individual has oligozoospermia)

Question 12

What 2 things does choice of method for measuring concentration of sperms depend on?

Answer 12

1. How dense or sparse the sample is
2. Dilution factor - small sample need to be diluted in water to immobilise sperms to be able to count them

Question 13

What determines how many grids to count

Answer 13

Dilution factor
1 in 20 dilution - tells you how many grids you count and this is written in the WHO

Question 14

Why should sperm motility be done ASAP after liquefection?

Answer 14

• semen motility should be assessed ASAP after liquefection of the sample, within 1 hour following ejeculation, to limit deleterious effects of dehydration, pH or changes in temp on motility

Question 14

How do you perform examination of sperm motility in the lab?

Answer 14

1. mix the semen well
2. Remove aliquots of semen immediately after mixing (~ 10 micrometers each), allowing no time for spermatozoa to settle in suspension
3. make a wet preparation approximately 20 micrometer deep (2 replica), wait for the sample to stop drifting(within 60 seconds)
4. examine the slide with phase contract optics at x200 or x400 magnification, assess approx 200 spermatozoa per replicate for percentage of different motile categories
5. compare the replica values to check if they are acceptably close, if so proceed with calculations; if not prepare new sample.

Question 15

What are 4 categories of sperm motility according to WHO?

Answer 15

1. rapidly progressive motility(a)
2. slowly progressive motility(b)
   c) non- progressive motility
   d) immotility: no movement

Question 16

a) Rapid progressive motiltiy

Answer 16

spermatozoa moving actively, either linearly or in a large circle, covering a distance from starting point to the end point, of at least 25 micrometer (or half tail length) in one second.

Question 17

b) slowly progressive motility

Answer 17

: spermatozoa moving actively, either linearly or in a large circle covering a distance, from the starting point to end points, of 5 to <25 micrometer (or at least one head length to less than 1/2 tail length) in one second

Question 18

c) non - progressive motility

Answer 18

: all other patterns of motiltiy with an absence of progression e.g. swimming in small circles, the flaggellar force hardly displacing the head, or when only flaggellar beat can be observed

Question 19

What is the ref value of a+ b according to WHO?

Answer 19

a+b >/= 30%
a+b+ c >/= 42%

Question 20

What is sample with motility below ref value called?

Answer 20

• Asthenozoospermia

Question 21

how was sperm motility previously classified?

Answer 21

1. progressive(a) - now further categorised into rapid and slow
2. non - progressive (b)
3. immotility (c)
   => previous ref values a>/= 32% and a+b >/= 40%

Question 22

sperm motility machine

Answer 22

Assign each button for each category , a, b, c, d
and each has a field of view
if you dont count them on time they may be lost from field of vision.
total is talling up at the end, when you get to 100 a beep sound and you have to press end button to release it.
=> new machine you dont need to convert raw counted values into percentage the machine does it for you automatically

Question 23

what are criteria of semen analysis?

Answer 23

• count only spermatozoa with head and tail intact
  -evaluate at least 200 spermatozoa in a total of at least 5 fields per replicate.
• Avoid repeatedly viewing the same field
• stick with the same region in all 5 fields, you can divide it into quadrats

Question 24

Assessments of sperm morphology

Answer 24

1. assessed directly on the wet preparation 2. using stains

Question 25

What are 3 common stains used?

Answer 25

1. papanicolaou staining 2. Diff-quick staining - based on HE stains 3. shorr stain

Question 26

Why is assessing directly on wet preparation preferred over using stains?

Answer 26

- stains can be toxic to sperms, eggs and embryos present in the IVF labs in treatment centres.

Question 27

What is normal sperm morphology- sperm head according to WHO 2010?

Answer 27

- smooth, regularly contoured and generally oval in shape - well - defined acrosomal region comprising 40-70% of the head area - Acrosomal region should contain no large vacules, and not more than two small vacuoles, which should not occupy more than 20% of the sperm head - The post -acrosomal region (where genetic info found) should not contain any vacuoles. - mid piece should be roughly the same size as sperm head and tail is much longer

Question 28

morphology below ref value is called ...

Answer 28

Tetratozoospermia

Question 29

What is sperm vitality?

Answer 29

- how many sperm cells are alive - dead sperm cells have damaged membrane so integrity of spermatozoa membrane is how you check for vitality using stains

Question 30

What are 2 methods of testing for sperm vitality?

Answer 30

1. Eosin - Nigrosin stain - membrane permeable in dead sperms (no vitality) so take in dye 2. hypo-osmotic swelling test - loop shaped tails of sperms as they take in solution

Question 31

vitality below ref value is ...

Answer 31

necrozoospermia

Question 32

Sperm pH

Answer 32

- reflects the balance between the pH values of the different accessory gland secretions, mainly the alkaline seminal vesicular secretion and the acidic prostatic secretion - assessed asap after liquefaction of the semen sample, preferably at 30 mins but no later than an hour post ejaculation.

Question 32

Leucocytes

Answer 32

- increased presence of leucocytes could be indicative of an infection - round cell counts using counting chamber - immunocytochemical staining

Question 33

summary

Answer 33

- a semen analysis is usually the first diagnostic step in male fertility investigations. - it is the analysis of seminal fluid and sperm parameters as an indicator of male fertility potential - a standard semen analysis is carried out using the WHO (2010) parameters for the lab assessment of human semen - sperm parameters analysed include: volume, liquefaction, appearance, concentration, motility, morphology, vitality, pH, leucocytes. All of these parameters are of clinical significance to male infertility investigation.