Semenology Update in Assisted Reproductive Technology discusses sperm samples, semen analysis, and sperm preparation techniques for IVF and ICSI. It outlines the different origins of sperm including ejaculate, epididymal, and testicular sources. Basic semen parameters like concentration, motility, and morphology are evaluated. Sperm preparation is necessary to isolate motile sperm, eliminate debris and dead sperm, and prepare functional sperm for ART procedures. Common techniques include swim-up, density gradient centrifugation, and direct centrifugation depending on sperm quality. The prepared sperm fraction is critically evaluated for concentration and motility before use in IVF or ICSI.

1. Semenology Update in Assisted
Reproductive Technology
• Abimibola Nanna
• Msc Clinical Embryology, UK

2. Learning Objective
• Know the different origins and types of sperm
samples
• Know the different parameters of basic semen
analysis
• Understand why sperm preparation is
necessary in ART
• Know the different techniques for sperm
preparation

3. Condt
• Understand the different types of sperm
samples for IVF or ICSI
• Evaluate the semen samples prior to IVF or
ICSI

4. Outline of Presentation
• Origin of spermatozoa
• The ejaculate basic semen parameters
• Why is semen preparation necessary
• Different preparation techniques
• Method and procedure for preparation for
different types of semen samples
• Critical evaluation of the prepared semen fraction
for IVF or ICSI
• Sperm preparation conclusion

5. Origin of Spermatozoa
Ejaculate origin:
• Natural, retrograde , or electro-ejaculate
• Frozen ejaculate, coitus with non- spermicide
condon
Epididymal or Testicular origin
• Testicular sperm extraction-TESE
• Microsurgical epididymal sperm aspiration-
MESA

6. Cond
Percutaneous epididymal/ testicular
extraction
• Fine needle aspiration from testis-TESA
• Percutaneous epididymal sperm aspiration-
PESA

7. The Ejaculate
• Lower limit for semen volume is 1.5ml
• Ejaculate consist of 10% cells and 90% liquid
• The cell fraction consist of : spermatozoa, rounds,
other debris
• The liquid consist of : prostate fraction, seminal vesicle
fraction, minor fraction from bulbourethral and
epididymides
• The quality of the ejaculate is determine by: testes,
accessory organ secretion, abstention and illness
• Abstinence period of 2-3 days before ejaculation and
maximize conception rate

8. Basic semen parameters in ART
• Concentration
• Motility
• Morphology

9. Sperm concentration-2010 W.H.O
• Concentration: number of spermatozoa per ml
• Concentration lower reference limit is 15
million/ml
• Total sperm count lower reference limit is 39
million per ejaculate
• Recommended counting chamber is 100um
deep haemocytometer or Makler chamber
• Concentration can fluctuate due to
environmental and non-environmental factors

10. Cond
• Polyzoospermia, oligozoospermia,
cryptozoospermia, necrozoospermia and
azoospermia

11. Motility
• Expressed in percentage of spermatozoa
according to W.H.H 2010 guidelines
• Accessed as soon as possible, within one hour of
production using wet preparation
• Progressive motility-PR
• Non-progressive motility-NR
• Immotile-IM
• Progressive motility normal value of 32%
• Total progressive motility (PR+NR)-40%

12. Cond
• Asthenozoospermia, necrozoospermia
• Progressive motility is crucial for IVF, IUI

13. Sperm Morphology 1
• Morphological assessment is done through the
staining method: papanicolaou stain, Shorr and
Diff Quik stain
• Categories of defects:
 Head: tapered, pyriform, round, large,
acrosome abnormalities, vacuoles, amorphous,
double head
 Neck and midpiece broken, irregular , thin,
cytoplasmic droplet.
 Tail: short, coil and multiple, broken or bent

14. Sperm morphology 2

15. Sperm morphology 3
• Normal categories:
Head: oval in shape with 40-70% acrosomal
region of the head and 4.1um long
Midpiece: slender , regular and 4um long
Principal piece: thinner than midpiece and
approximately 45um long

16. Sperm morphology 4

17. Sperm morphology 5
• Normal values for sperm morphology is ≥4%
• Teratozoospermia and globozoospermia
• Can be affected with the days of abstinence
• Can be affected by the environment

18. Why sperm preparation
• Capacitation:
Separation of spermatozoa and seminal plasma
Biochemical process of plasma membrane-
hyperactivation
 Preparation for acrosome reaction
• Isolation of subpopulation of spermatozoa with
progressive motility and normal sperm morphology
• Elimination of dead spermatozoa , other
contaminating cells and debris

19. Contd
• Elimination/reduction of leukocytes
• Elimination/reduction of bacteria
• Elimination/reduction of reactive oxygen
species
• Enrichment of functional sperms with good
DNA integrity

20. Sperm preparation
• Depends on sperm concentration
• Normal count and motility-swim up
• Subnormal semen parameters- density
gradient
• Severe oligozoospermic- direct centrifugation

21. Direct Swim up
• Spermatozoa are allow to swim up into a fraction
of culture medium
• Upper section of the overlaying culture which
contain motile cells is removed and centrifuged
• Exclusion of debris, immotile spermatozoa and
other cells
• Pellet is re-suspended in culture medium
• Swim up techniques has variation in the type and
volume of overlaying medium
• Round bottom tubes are used for this separation

22. Swim up 2

23. Advantage
• For normal sperm concentration and motility
• Elimination of reactive oxygen species
• Excellent pregnancy rate for IUI, IVF procedure
• Sperms cell with high level of DNA integrity
• Selection of sperm cells with intact membrane
and morphology
• Cost-effectiveness-cheap

24. Disadvantage
• Lower recovery of motile spermatozoa
• Time consuming

25. Density gradient 1

26. Density gradient 2
• Principle: Centrifugation through a gradient with
different densities (weight/volume)
- Two step discontinous gradient
- Laying isotonic suspension with different
concentration of collodial silica particles coated
with silane.
-Highest density fraction on the bottom of the tube
-Semen fraction on top of the gradient
-More than one test tube can use for oligozoospermic
and asthenozoospermic samples

27. Density gradient 3
• Centrifugation of the gradient: Distribution of
sperm according to their density
-Highest motile spermatozoa harvested from
the bottom and overlay with 0.5ml of sperm
prep medium
-The best fraction for fertilization
-Immotile spermatozoa, other cells and debris
are retained in the low density fraction

28. Advantage
• Numbers of density layers can be two or three
• Lower production of reactive oxygen species
• Less chromatin defects obtained
• Valid for sperm concentration that is great
than 4million/ml
• High recovery of motile spermatozoa that is
free from debris, leukocytes and germ cells
• Easy to standardized

29. Disadvantage
• Round cells concentrate at the interface
between two layers
• Creation of barriers that is impermeable to
spermatozoa with round cells sample
• Low recovery rate of spermatozoa with round
cells samples
• It cannot be use for severe oligozoospermia as
spermatozoa will be lost at the low density
fraction

30. Disadvantage
• Lower DNA integrity
• Production of good interphases between
layers can take some time
• Multiple centrifugation

31. Centrifugation method for severe
oligozoospermia
• Centrifuge raw sperm with sperm preparation
medium at 1800g for 5 minutes
• Remove supernatant and add small volume of
sperm prep medium over the pellet
• Allow it to undergo swim up and aspirate the
upper layer that contain motile sperm cells fpr
ICSI

32. Sperm preparation 1
• Fresh ejaculated semen
- semen collection done near the lab
-Reception of sample and patient identification
- Liquefaction
- Evaluation of semen parameters according to
W.H.O criteria
• Preparation techniques; density gradient or swim up
• Preparation of the selected fraction for IVF or ICSI by
adding 0.5 culture medium
• Evaluation of final fraction concentration and motility

33. Sperm preparation 2
• Retrograde ejaculated semen
-Semen passes into the bladder at ejaculation
-Sodium bicarbonate intake for the alkalinization of the
bladder
• Three fraction are collected and examined
-Antegrade fraction
- First retrograde urine fraction in buffer medium
- Remaining of retrograde urine fraction
-Examination of these fractions
• Washing and concentration of retrograde fraction
• Preparation for ICSI is similar to fresh ejaculate

34. Sperm preparation 3
• Frozen ejaculated semen
-Partner sperm of donor sperm
-Number of straws for thawing depends on
indication; sperm quality and fertility treatment
-Identify double check for all straws
-Disinfection of straws
-Preparation is similar to fresh ejaculated samples
-Warning: avoid osmotic shock by dropwise
addition of washing medium to frozen semen
samples from the sperm bank

35. Sperm preparation 4
• Open testicular sperm extraction
-For non-obstructive azoospermia
-Local anesthesia
-Collection of biopsy sample-seminiferous
tubules into buffer
-Mechanical shredding of biopsy samples
under stereomicroscope with sterile needles
or scissors to release spermatozoa from tubuli

36. Sperm preparation 4
-Put the product into a 5ml falcon tube and
centrifuge for 5 minutes at 1800g.
-Remove supernatant with a pasteur pipette
and suspend pellet in 0.2ml of culture
medium
-Retrieve sperm cell from pellet through swim
up
-For retrieval of sufficient sperm cells,
incubation is done for 24 hours

37. Sperm preparation 4
- Biopsy collection should be done a day
before oocyte retrieval
-When immotile spermatozoa, spermatozoa
are attached to other tissues or no
spermatozoa is seen, then enzymatic digestion
of tissues and erythrocyte lysis should be
done

38. Sperm preparation 5
• Microsurgical epididymal sperm aspiration MESA
-For obstructive azoospermia patients
-It is a blind procedure where a cut is made in
a single tubule of the epididymis and the content
is aspirated
-Aspirates are released in buffered medium
-Preparation by density gradient
centrifugation
-Sample can be frozen for future use

39. Sperm preparation 5
• Percutaneous epididymal sperm aspiration PESA
-For obstructive azoospermia patient
-It is a blind procedure where a fine needle is
used to puncture the epididymis
-Sample is aspirated percutaneously
-Aspirates are released in buffer medium
-Similar preparation procedure as for MESA

40. Sperm preparation 5
• Testicular sperm aspiration- TESA
-For obstructive azoospermia
-it is done where there is no sperm cells in the
epididymis
- It is a blind procedure under local anesthesis
- A wide bore needle is used to aspirate
sperm cells directly from the testis
- Petri dish with micro droplets of buffer
medium

41. Sperm preparation 5
-Testicular aspirates are released in the
droplets
-Check for motile sperm cells
-Repeated aspiration can be done
-No freezing is possible for spermatozoa in
droplet under oil

42. Critical evaluation of the fraction for
IVF
• Embryologist critically evaluates the final fraction for
IVF
• Motility: Final fraction with 100% progressive motility
• Concentration: Progressive motile sperm concentration
is between 1-20million/ml after sperm preparation
• Sperm volume to be added to each insemination
well/droplet is calculated
• 100,000 progressive motile sperm cells per oocyte in a
microdroplet under oil or each well four well dish.

43. Critical evaluation of the fraction for
ICSI
• The least sluggish motility separate viable
from dead spermatozoa
• Immotile spermatozoa:
- Demand for second semen sample
-Hypo-osmotic swelling test for sperm cell
selection.
• Hyaluronic acid binding method

44. Conclusion
• Basic seminal parameters like comcentration,
motility are assessed in ART
• Different sources of spermatozoa are
examined in ART and extracted
• Spermatozoa are separated from seminal fluid
by direct swim up or density gradient to
obtain functional spermatozoa for ART.
• IVF is for normal semen parameters while ICSI
for abnormal semen parameters

45. THANK YOU