Sperm Assesmen&preparation&cryopreservation.ppt

Gyn

2. Eman Anwar Hassan Mohamed
Assis.prof. of embryology (IICPSR) Al-Azhar University
IVF Lab director in ART unit Al-Azhar University
IVF Lab director in Walad we Bent IVF center

4. Complete semen analysis
a-Sample collection:
Semen samples are collected by masturbation after
2 to 7 day period of abstinence , long period of abstinence (10
day) lead to decreased motility and shorter period result in
low volume and density.
The container must be clean, sterile and
wide mouthed to minimize collection.
The semen specimen should be
maintained at room or body temperature
and examined within 1 hour of collection.

5. B-Physical examination
1-Semen volume: The method of measuring semen
volume is by drawing the entire ejaculate up into a sterile
(and warmed) graduated glass pipette .
2 -Color:A normal semen sample has a homogenous, ray
opalescent appearance. The semen pink or even reddish if
blood is fresh, while old blood will color the semen a
brownish color.

6. 3-Odor: The semen odor may be affected by some foods
or drugs excreted in semen. It may be offensive in some cases of
genital infection or urineferous in cases of semen contamination by
urine .
4-Liquefaction: A normal semen sample liquefies within 30 minutes at
room temperature, although, this usually occurs within 15 minutes.
In some cases, complete liquefaction does not occur within 60 minutes,
and this should be recorded. Normal semen samples may contain
jelly-like grains (gelatinous bodies) which do not liquefy and do not
appear to have any clinical significance.

7. C-Microscopic examination
Microscopic examination includes evaluation of sperm
concentration, motility, morphology and the presence of
cellular elements other than spermatozoa.

8. 1-Sperm concentration:
Haemocytometer chamber Makler Chamber Computer Assisted SA (CASA)

9. 2-Sperm motility
A drop of well mixed undiluted semen is placed on the
surface of warm, dry and clean microscopic slide and then
cover slip is placed. The slide then allow to rest on the
bench or on the microscopic stage until stoppage of fluid
movement. The drop of semen is then examined at
magnification of 400 x.
Both the motile and immotile sperms are counted in at least 5
separate microscopic fields, at minimum 200 spermatozoa
should be assessed .The percentage motility is calculated
from the mean value.

10. Categories of sperm movement
1-Progressive motility (PR): spermatozoa moving actively,
2-Non-progressive motility (NP): all other patterns of
motility with an absence of progression
3-Immotility (IM): no movement (WHO,2010).

11. Sperm vitality
Vitality test using hypo-osmotic swelling:-
when choosing spermatozoa for ICSI; spermatozoa with intact
membranes swell within 5 minutes in hypo-osmotic medium and all
flagellar shapes are stabilized by 30 minutes
dilute the medium to be used 1 + 1 (1:2) with sterile, purified water.
5 minutes incubation when spermatozoa are to be processed
Scoring
1. Swollen spermatozoa (Vital-Live) are identified by changes in the
shape of the cell, as indicated by coiling of the tail

13. 2-Sperm morphology:
Sperm morphology evaluation according to strict criteria
uses a holistic approach, starting with the preparation of
clean microscope slides, the correct preparation of thin
semen smear and evaluation of slides.

14. Difference between sperm agglutination and sperm aggregation

16. Morphological abnormalities of sperm
•1-Headdefects:
Large, small, tapered, pyriform, round and amorphous head,
vacuolated head , head with small acrosomal area (< 40% head
area) and double heads or any combinations of these .
•2-Neck and midpiece defects:
Bent neck, bent mid piece, asymmetrical insertion into the
head, irregular, thick, and thin or any combination of these.
•3-Tail defects:
Long, short, multiple, hairpin, broken tail, bent tail, tails of
irregular width, coiled tail or any combination of these.

17. 4-Cytoplasmic droplets:
Morphologically normal spermatozoa must have an amount of
cytoplasmic material that does not exceed the size of a normal
sperm head by >30%.The cytoplasmic material is usually
situated at the base of the sperm head.
5-Tail Stump Syndrome
The tail stump syndrome is characterized by flagella organized
as uniflagellates and extremely short axoneme with 9 + 2 or 9
+ 0 arrangement generally with dynein arms. In contrast, the
short tail syndrome includes biflagellate arrangement, 9 + 0 or
9 + 1 axoneme and lack of dynein arms. In both syndromes,
spermatozoa show usually unaffected sperm heads but
unassembled mitochondria(Kruger, 2004).

18. A-Rounded head B -Triple head. C-Pinucleated head.
D-Bent neck. E-Thin midpiece. F-Thick midpiece.
G-Double tail. H-Triple tail. I -Tail Stump Syndrome.

19. •A-Genetically determined sperm defects:
•1-Globozoospermia
•2-Short tail syndrome
•

21. •Terminologies of SA
Oligospermia : sperm concentration <15 million/ml
Asthenozoospermia :<40% grade (PR+NP) or < 32 PR%
Teratozoospermia : <4% spermatozoa
OAT =Oligo-astheno-teratozoospermia
Azoospermia :no spermatozoa in semen
Polyzoospermia : high sperm concentration, >200M/ml
Hypospermia : semen volume < 1.5 ml
Hyperspermia : semen volume > 6.0 ml:
Aspermia : no semen volume
Pyospermia :leukocytes present in semen, >1M/ml
Hematospermia :red blood cell present in semen
Necrozoospermia :“dead” sperm

23. Sperm preparation
The semen is a mixture of motile and dead spermatozoa with
cells, cellular debris and sometimes micro-organisms present.
A variety of methods have been developed to separate the motile
sperms from the ejaculate. The most common methods are
washing and centrifugation .
1-Simple sperm wash
2-Swim up
3-Sperm Gradient
All preparations should done in a laminar flow for sterility.

24. Sperm preparation techniques for A.R.T.
Two routine preparation techniques are described:
-swim-up
-Discontinuous density gradients
1-Swim-up
add 2 ml of Ham-F 10 medium on fresh sample and centrifuged at 1000 rpm for 10
minutes, supernatant removed and the sperm in the resulting pellet were allowed to
swim up in 1 ml Ham-F10 overlay for 30 mints, , the number of motile and
morphologically normal sperm cells needed for assisted reproduction..
Before after

25. 2-Discontinuous density gradients
This technique gives good results in many cases.
Alternative products (AP) replace Percoll*which has been with
drawn from use in human clinical applications
The technique uses alternative products in three layers :
1-lower layer : AP 90 % conc.
2-intermediary layer : AP 45 % conc.
3-Upper layer : patient sperm

26. After centrifugation, Two upper layers are eliminated and the pellet at the
bottom of the AP 90 % is resuspended in 1 ml specific medium .
a swim-up procedure for having a best sample.

27. Preparing HIV-infected semen samples
If the human immunodeficiency virus (HIV) is present in semen, viral
RNA and proviralDNA can be found free in seminal plasma and in
non-sperm cells
A combination of density-gradient centrifugation followed by
swim-up has been proposed as a way of preventing infection
of uninfected female partners

28. Preparing retrograde ejaculation samples
Alkalinizationof the urine by ingestion of sodium
bicarbonate, for example, will increase the chance that any
spermatozoa passing into the urine will retain their motility
characteristics

29. In case of Azoospermia
Sperm Retrieval Procedures
In some semen samples there may be no sperm present in
the ejaculate. There can be various reasons for an absence
of sperm known as Azoospermia.


30. PESA
Percutaneous epididymal sperm aspirationis a procedure
performed for men who are having sperm retrieved for in
vitro fertilization/intracytoplasmic sperm injection
(IVF/ICSI) who have obstructive azoospermia .It is done
with local anesthesia in the operating room

31. TESA
Testicular sperm aspiration (TESA)is a procedure
performed for men who are having sperm retrieved for
sperm injection (ICSI).It is done with local anesthesia in
the operating room .A needle is inserted in the testicle
and tissue/sperm are aspirated.
TESA is performed for men with obstructive azoospermia
and often doesn’t provide enough tissue/sperm and an
open testis biopsy is needed.

32. TESE
Testicular sperm extraction are procedures performed for men
who have testis failure.The procedure is performed to see if
there are sperm present as well as for pathologic diagnosis to
evaluate for malignancy.
TESE is usuallyperformed
in the operating room.
Patients usually
cryopreserve sperm during
this procedure for future
IVF/ICSI.

34. Sperm freezing:
Semen samples are slowly frozen in liquid nitrogen
vapors. Once the samples have been frozen in the
liquid nitrogen vapors, they are placed in special
containers where they are stored in the liquid nitrogen
until they are needed .