1. Semen analysis & Sperm processing
Jalal Ghasemzadeh
Andrology lab

2. What is Andrology?
“Andro” from the Greek “andros”, man.
Andrology is the study of male fertility and laboratory aspects of infertility diagnosis and treatments.
Science of diseases of males, including infertility, spermatogenesis and sexual dysfunction.

3. Semen analysis Alternative names : sperm test male infertility test
Semen analysis (SA) is a gateway test and primary test for evaluation of male infertility and treatment planning for helping couples achieve pregnancy.
Safe, inexpensive, non-invasive procedure
Alternative names :
sperm count
sperm test
male infertility test
spermogeram

4. Indications of semen analysis
1) Assessment of fertility (2 sample,7days to 3weeks)
Phosphatase acid
2) Forensic purposes PSA
Sperm
3) Suitability for artificial insemination (IUI, IVF)
4) Post vasectomy (2 sample, 3 months and 6 months )

5. Semen source Characteristics Volume Source of secretion
alkaline & gelatinous fructose, prostaglandins seminogelin
1-3 cc (46-80%)
Seminal vesicles
acidic & watery contain zinc, citric acid, acid phosphatase & PSA
0.5-1 cc (13-33%)
Prostate
contain spermatozoa , carnitine & inhibin
cc (5%)
Testis,epididymids & vasa deferens
viscous & clear
contain IgA & Mucoproteins
cc (2-5%)
Bulbourethral glands

6. Male reproductive system
Erectile tissue
Prostate gland
Urinary bladder
Bulbourethral gland
Vas deferens
Epididymis
Testis
Seminal
vesicle
(behind bladder )
Urethra
Scrotum
Glans penis
Erectile tissue
Prostate gland
Urinary bladder
Bulbourethral gland
Vas deferens
Epididymis
Testis
Seminal
vesicle
(behind bladder )
Urethra
Scrotum
Glans penis

7. Accessory glands dysfunction
1) Seminal vesicles dysfunction :
Low volume
Decreased PH
Decreased fructose concentration
2) Prostate dysfunction :
Delayed liquefaction
Increased viscosity
Increased PH
Decreased zinc concentration

8. Spermatogenesis Begins at puberty and continues throughout
adult life of a male.
Sperm are produced within the seminiferous
tubules.
Sertoli cells (nurse cells) feed and protect
from a blood-testis barrier developing sperms.
Primordial germ cells differentiate into
spermatogonia (diploid cells).
Spermatogonia during mitosis division become
two primary spermatocyts.

9. Spermatogenesis Each primary spermatocytes (diploid cells )
through meiosis I produce two secondary
spermatocytes (haploid cells).
Each secondary spermatocytes during
meiosis II divides to produced two spermatid
(haploid cells with 23 chromosomes).
As a result of the two meiotic divisions, each
primary spermatocyte produces four spermatids.

10. Spermiogenesis Spermatogenesis takes 64 days in the human.
Changes that transform
spermatids into sperm :
1) Chromatin condensation
2) Flagellum formation
3) Acrosome cap development
4) Discarding excess cytoplasm
Sperms transported to the epididymis
(caput corpus caudal ) here they are stored.
Spermatogenesis takes 64 days in the human.

11. Examinations of human semen
1) Standard procedures
Sample collection & delivery
Initial microscopic & macroscopic examinations
Anti-sperm antibody test (IBT & MAR test)
2) Optional tests
Indices of multiple defect (TZI & SDI )
HOS test
Semen culture
CASA
Biochemical assay (fructose, zinc)
3) Research tests
Sperm function tests
ROS
Acrosome reaction
CASA morphology

12. Sample collection & Delivery
1) Two samples days……3weeks
2) Sexual abstinence days……7days
3) Adequate collection
masturbation
non toxic glass or plastic container
no condoms
no lubricants
complete (important)
4) Adequate lab delivery ≤1 hour post collection
5) Adequate temperature C – 40 0C

13. Long sexual abstinence (Levitas et al. 2005) & (Pellestor et al.1994)
Sample collection
Long sexual abstinence (Levitas et al. 2005) & (Pellestor et al.1994)
Increase sperm concentration
Increase volume
Decrease motility
Decrease normal morphology
Loss of the first part of the ejaculate :
Increase PH
Decrease sperm concentration
Delay liquefy
Loss of the second part of the ejaculate :
Decrease PH
Decrease volume
Lack of coagulum formation

14. Sample collection at clinic or at home, which‼?
Elzanaty & Malm, 2007
OBJECTIVE : Comparison of sperm parameters in samples collected by masturbation at
clinic and at home.
at a clinic (n = 273)
PATIENTS men
at a home (n = 106)
RESULTS :
1) Sperm concentration, total sperm count & rapid
progressive motility were statistically significantly
higher at home-collected samples.
2) Semen volume, normal morphology, PSA, Zinc &
fructose did not differ significantly between groups.
CONCLUSION : Superior semen quality in samples collected by masturbation at home compared
with at a clinic. this should be taken into consideration in infertility investigation.
Home
Clinic
oligozoospermia
19/106 (18%)
81/273 (30%)
asthenozoospermia
68/106 (64%)
205/273 (75%)

15. Semen collection methods
1) Masturbation
2) Coitus interruptus :
Loss of first portion of the ejaculate
Cellular & bacteriological contamination
The acid PH of the vaginal fluid
3) Semen collection in sexual dysfunction
Vibrator
Electro ejaculator (E.E)
Sperm retrieval methods :
PESA (Precutaneous Epididymal Sperm Aspiration)
MESA (Microsurgical Epididymal Sperm Aspiration)
TESE (TEsticular Sperm Extraction)
MESA
PESA

16. Initial examinations 1) Macroscopic : 2) Microscopic : Liquefaction
Appearance
Viscosity PH
Volume
2) Microscopic :
Sperm motility
Sperm viability
Sperm concentration
Sperm morphology
Cellular elements other than sperm

17. Macroscopic examinations
1) Liquefaction time
Definition : the change from the coagulated to liquid.
Principl : proteolysis semenogelin by PSA
Normal : within 60 minutes bromelain(1g/l)
Abnormal : >60 minutes after ejaculation alpha amylase
chymotripsin(150 USP/ml)
2) Appearance
Normal: homogenous & grey-opalescent
Abnormal:
Opaque (debris or WBC)
Red −Brown (RBC)
Yellow (jaundice or taking vitamins)
Clear (poor sperm quantity)

18. Macroscopic examinations- continued
3) Viscosity (consistency)
Semen viscosity refers to the fluid nature
↑ Viscosity = ↓ sperm motility
Normal: ≤ 2 cm thread
4) PH
Normal = 7.2 or more
PH is important because sperm die at PH < 6
Routine measurement of PH is not necessary.

19. Macroscopic examinations- continued
5) Volume
 Low volume (hypospermia) : < 0.5ml
Seminal vesicle dysfunction or agenesis
Ejaculatory ducts obstruction
Retrograde ejaculation
Incomplete collection
High volume (hyperspermia) : > 6ml
Long periods of sexual abstinence
Over production of accessory glands
No ejaculate (Aspermia) :
Surgery

20. Assessment of motility
Microscopic examinations
CASA ( Computer- Aided/Assisted Sperm Analysis )
 Systematic (manual method) :
Grade a → rapid progressive motility ( ≥25 µm/s at 37 0C )
Grade b → slow progressive motility ( 5-25µm/s at 37 0C)
Grade c → non progressive motility (<5 µm/s at 37 0C)
Grade d → immotility
Asthenozoospermia:
Sperm structure defects
Prolonged abstinence periods
Varicocele
Anti-sperm antibody
Cartagena's syndrome
Necrozoospermia : all sperms are dead
Cilia immotile syndrome

21. Microscopic examinations- continued
Viability
Microscopic examinations- continued
Distinguish live non motile sperm from dead.
Indication > 50% immotile spermatozoa
Determined by :
 Eosin staining
 Eosin- nigrosine staining
 Hypo-osmotic swelling test (HOS test )
Hoechst staining
Live
Dead
Live
Dead

22. Microscopic examinations- continued
Sperm concentration
Sperm concentration = number of sperm per ml of semen.
Azoospermia (no sperm in the ejaculate)
Obstruction
Hormonal insufficiency
Congenital Klinefelter's syndrome
Oligozoospermia ( <20 million sperm/ml )
Procedural causes
Varicocele
Retrograd ejaculation
Polyzoospermai ( >250 million sperm/ml )
Long sexual abstinence
Improved neubauer
Makler counting chamber

23. Assessment of morphology
Microscopic examinations- continued
Most confusing & time-consuming area of semen analysis
Smearing, air-drying, fixation, staining
Staining method :
 Papanicolaou stain→ method recommended
 Pap-quick stain
 Diff-quick stain→ rapid staining method
 Shorr stain
 Wright-gimsa stain

24. Structure of normal spermatozoa
1) Head nuclear region
Oval in shape
Length: 4-5 µm & Width: µm
Length-to-width ratio: 1.5 to 1.75 µm
Acrosomal region: 40-70% head area
Contain nucleus (DNA)
2) Midpiece metabolic region
Slender & Width <1 µm
Length: about 1.5 times the length of the head
Contain axoneme & mitochondria
Cytoplasmic droplets less than half the size of the normal head
3) Tail locomotor region
Straight, uniform, thinner than the midpiece, uncoiled
Length: approximately 45 µm

25. Classification of sperm morphology
1) Head defects
Small
Large
Tapered
Round
Amorphous
Vacuolated
Pyriform
2) Neck & midpiece defects
Bent
Thin
Thick
Asymmetrical insertion
3) Tail defects
Short
Coiled
Double
4) cytoplasmic droplet or excess residual cytoplasmic ( >1/3rd normal head )

26. Diff- quick staining (x100)

27. Scanning Electron Microscope of spermatozoa

28. Sperm morphology classification systems
Normal reference range
1) Macleod >60%
2) WHO manual 2nd edition >50%
3) WHO manual 3rd edition >30%
4) Strict (menkveld & kruger ) / WHO manual 4th edition >14%
5) WHO manual 5th edition >4%
6) ASCP (American society clinical pathology ) >80%

29. Cellular elements other than spermatozoa
Microscopic examinations- continued
Cellular elements other than spermatozoa
Round cells : < 5x106/ml
Immature germ cells :
Spermatogenic cells : fever, radiation, cytotoxic drug
Prostatic cells
Epithelial cells
Leukocytes :
Leukocytospermia ( >1x106/ml )
Pyospermia (high amount of WBC)
Produce ROS, motility, aggregation
Antibiotic treatment

30. Reference values of semen variables (WHO 1999)
2.0 ml or more
Volume
7.2 or more pH
20x106 spermatozoa/ml or more
Sperm concentration
40x106 spermatozoa per ejaculate or more
Total sperm count
50% or more motile (grades a+b ) or
25% or more with progressive motility (grade a )
Motility
30% or more with normal morphology
Morphology
50% or more live i.e. excluding dye
Viability
Fewer than 1x106/ml
White blood cells
Fewer than 50% motile spermatozoa with adherent particles
MAR test

31. Lower reference limits for semen (WHO 2010)