Diagnostic Assessment Flashcards
What is the first diagnostic step in male fertility investigation?
-analysis of seminal fluid and sperm parameters as an indicator of male fertility potential using WHO criteria for normal semen parameters (2021)
What are different types of semen analysis?
- manual semen analysis (preferred in clinical setting)
- computer- assisted semen analysis (CASA)
How is manual semen analysis carried out?
- lab practitioner uses light microscope for cell count
-used more inc clinical setting
How does CASA work?
- 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
WHO reference values (2021)
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
Assessment of appearance
- grey- opalescent appearance
what are 2 methods of measuring sperm volume?
- Direct volume measurement method :
- volume (ml) measured directly using a serological pipette - 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
Assessment of liquefaction
-normal liquefaction should take place within 20-30 minutes post production (abnormally long liquefaction time may be indicative of an infection eg. bacterial prostatis)
What are 2 devices used to measure sperm concentration?
- Neubauer haemocytometer - the one we use, grids and look at dilution factor.
- Makler counting chamber
Assessment of concentration
sperm conc = quantity of sperm present in a sample
- measured in millions per ml
- determined using counting chamber
What are samples below the reference value concentration known as?
-oligozoospermia (individual has oligozoospermia)
What 2 things does choice of method for measuring concentration of sperms depend on?
- How dense or sparse the sample is
- Dilution factor - small sample need to be diluted in water to immobilise sperms to be able to count them
What determines how many grids to count
Dilution factor
1 in 20 dilution - tells you how many grids you count and this is written in the WHO
Why should sperm motility be done ASAP after liquefection?
- 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
How do you perform examination of sperm motility in the lab?
- mix the semen well
- Remove aliquots of semen immediately after mixing (~ 10 micrometers each), allowing no time for spermatozoa to settle in suspension
- make a wet preparation approximately 20 micrometer deep (2 replica), wait for the sample to stop drifting(within 60 seconds)
- examine the slide with phase contract optics at x200 or x400 magnification, assess approx 200 spermatozoa per replicate for percentage of different motile categories
- compare the replica values to check if they are acceptably close, if so proceed with calculations; if not prepare new sample.
What are 4 categories of sperm motility according to WHO?
- rapidly progressive motility(a)
- slowly progressive motility(b)
c) non- progressive motility
d) immotility: no movement
a) Rapid progressive motiltiy
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.
b) slowly progressive motility
: 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
c) non - progressive motility
: 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
What is the ref value of a+ b according to WHO?
a+b >/= 30%
a+b+ c >/= 42%
What is sample with motility below ref value called?
- Asthenozoospermia
how was sperm motility previously classified?
- progressive(a) - now further categorised into rapid and slow
- non - progressive (b)
- immotility (c)
=> previous ref values a>/= 32% and a+b >/= 40%
sperm motility machine
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
what are criteria of semen analysis?
- 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
Assessments of sperm morphology
- assessed directly on the wet preparation
- using stains
What are 3 common stains used?
- papanicolaou staining
- Diff-quick staining - based on HE stains
- shorr stain
Why is assessing directly on wet preparation preferred over using stains?
- stains can be toxic to sperms, eggs and embryos present in the IVF labs in treatment centres.
What is normal sperm morphology- sperm head according to WHO 2010?
- 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
morphology below ref value is called …
Tetratozoospermia
What is sperm vitality?
- 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
What are 2 methods of testing for sperm vitality?
- Eosin - Nigrosin stain - membrane permeable in dead sperms (no vitality) so take in dye
- hypo-osmotic swelling test - loop shaped tails of sperms as they take in solution
vitality below ref value is …
necrozoospermia
Sperm pH
- 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.
Leucocytes
- increased presence of leucocytes could be indicative of an infection
- round cell counts using counting chamber
- immunocytochemical staining
summary
- 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.
