The document outlines the key procedures involved in ICSI lab work for a gynecologist, as presented by Aboubakr Elnashar from Benha University in Egypt. It discusses 8 main procedures: 1) semen preparation, 2) oocyte identification, 3) oocyte denudation, 4) oocyte assessment, 5) oocyte injection, 6) embryo selection, 7) embryo transfer, and 8) cryopreservation. For each procedure, it provides details on the aim, methods, and considerations. The document serves as a reference for best practices in ICSI lab work to optimize outcomes.

1. ICSI Lab
Procedures
For Gynecologist
Aboubakr Elnashar
Benha University, Egypt
ABOUBAKR ELNASHAR

2. Lab Procedures
1. Semen Preparation.
2. Oocyte Identification.
3. Oocyte Denudation.
4. Oocyte Assessment
5. Oocyte Injection.
6. Embryo Selection
7. Embryo Transfer.
8. Cryopservation.
ABOUBAKR ELNASHAR

3. qProcedures
ú Day 0
Sperm collection, and preparation
Oocyte retrieval
Insemination in vitro
ú Day 1
Check for fertilization (presence of 2 pronuclei or PN's)
ú Day 2
Embryos at the 4-cell or more stage of development
ú Day 3
Embryos at the 8-cell or more stage of development
ú Day 4
Embryos at the compacted morula (16-32 cell) stage
ú Day 5
Embryos at the blastocyst stage of development
ABOUBAKR ELNASHAR

4. 1. Semen Preparation.
q Obtaining the semen sample
1. An abstinence interval:
3-5 days.
2. Collection of the semen:
In the clinic is better than in the patient’s home.
3. Timing of processing of the semen:
With in 30 mins from ejaculation.
4. A frozen back-up sample should be requested
if sperm collection difficulty on the day of OR is anticipated.
ABOUBAKR ELNASHAR

5. qAim of Sperm preparation:
1. Eliminate:
seminal plasma, debris and contaminants
2. Concentrate:
progressively motile sperm
3. Select:
against morphologically abnormal sperm.
ABOUBAKR ELNASHAR

6. q Methods of Sperm preparation:
1. Discontinuous density gradient.
2. Double wash.
3. Swim up.
The swim-up technique and discontinuous density-
gradient centrifugation are most frequently used
and widely accepted.
ABOUBAKR ELNASHAR

7. qSelection of Sperm preparation method:
•Characteristics
•Origin of individual samples.
qIn absence of motile sperm.
-Phosphodiesterase inhibitors (pentoxifylline,
theophylline) or
-Hypo-osmotic swelling (HOS) test
ABOUBAKR ELNASHAR

8. qAzoospermia on the day of OR and in the absence
of a back-up sample:
•Sperm retrieval procedures or
•Oocyte cryopreservation
qIf no sperm are observed.
Enzymatic digestion of testicular tissue by
collagenase
ABOUBAKR ELNASHAR

9. 2. Oocyte identification
§ Aim:
Identify the oocytes from the aspirated follicular fluid.
oocytes are washed from the surrounding follicular
fluid (mimic physiology)
§ The time between OR and culture of washed
oocytes
should be minimal.
{Prolonged oocyte exposure to follicular fluid is not
recommended}
§ Exposure of oocytes to light
should be minimized.
ABOUBAKR ELNASHAR

10. 3. Oocyte Denudation
§ Chemical:
repeated pipetting in hylaundase enzyme.
§ Mechanical:
using thin pipettes of varying diameters (175-125 µ)
ABOUBAKR ELNASHAR

11. A and B: GV
C : MI
D : MII oocyte.
ABOUBAKR ELNASHAR

12. 4. Oocyte Assessment
qImportance of oocyte quality:
§ has a great influence on:
§ Fertilization
§ early development
§ implantation of embryos.
§ In order that the oocyte would be successfully
fertilized and cleaved, the nucleus and the
cytoplasm of the oocyte must be mature at the
same time.
ABOUBAKR ELNASHAR

13. qNuclear Maturation
1. Germinal Vesicle.
2. Metaphase 1 oocytes.
3. Metaphase 2 oocytes.
ABOUBAKR ELNASHAR

14. Oocyte maturity after cumulus and corona cell removal.
(A)Germinal vesicle (GV) stage oocyte is recognized by the
presence of a typical GV.
(B)Oocytes that have undergone GV breakdown but not yet
extruded the first polar body are called metaphase I oocytes.
(C)Metaphase II oocyte displays the presence of a first polar
body, which indicates that the oocyte is mature and has
reached the haploid state. Only metaphase II oocytes are
submitted to intracytoplasmic sperm injection.
ABOUBAKR ELNASHAR

15. 1. Germinal Vesicle
GV oocyte.
ABOUBAKR ELNASHAR

16. 2. Metaphase 1 oocyte
§ After GV breakdown oocytes reach metaphase I,
characterized by the absence of the first polar
body extrusion.
§ Though some of these intermediate eggs may
even be fertilizable
(De Vos et al., 1999),
ABOUBAKR ELNASHAR

17. qRetrieved immature oocytes after COH
ú Usually a variable percentage of the retrieved
oocytes from mature follicles after COH are GV or
M I.
ú These oocytes may change to M II oocytes in vitro,
and hence can be injected.
ú However, the reproductive outcome of these
oocytes is very poor.
ú This effect may not be evident until pre and early
postimplantation period.
ú This poor outcome can be explained by the
asynchronous nuclear and cytoplasmic maturity.
ABOUBAKR ELNASHAR

18. qFertilization and pregnancy potential of immature
oocytes from stimulated ICSI cycles.
(Shin et el , 2013)
§ The aim was to compare the outcome of retrieved
mature oocytes and retrieved immature oocytes that
were injected after maturation.
§ The fertilization rate of retrieved immature oocytes
was half that of retrieved mature oocytes (37% Vs
73%).
ABOUBAKR ELNASHAR

19. 3. Metaphase II oocyte
§ Large uniform cytoplasm
§ Discrete intact first polar body
ABOUBAKR ELNASHAR

20. Oocytes in first row represent different degrees of
vacuoles in cytoplasm.
Each oocytes in second row has increased central
granularity.
ABOUBAKR ELNASHAR

21. qCytoplasmic maturation
§ The only method:
oocyte fertilization
embryo development in vitro.
§ If the cytoplasm of oocytes is immature:
embryo cannot develop normally after
fertilization.
ABOUBAKR ELNASHAR

22. qDefects in Cytoplasmic maturation lead to:
1. Retarded embryos
2. Cleavage arrest
3. Failure of implantation
ABOUBAKR ELNASHAR

23. 5. Oocyte Injection
1. Sperm selection.
2. Sperm immobilization.
3. Oocyte adjustment:
polar body is at 12 or 6 o'clock.
4. Penetration of the oolemma
5. Aspiration of the cytoplasm.
6. Injection of the sperm.
ABOUBAKR ELNASHAR

24. (C) The injection pipette is introduced at the 3 o0clock position and rupture of
the oolemma is ascertained by slight suction. Then the sperm cell is delivered
into the oocyte with a minimal volume of medium; afterwards, the pipette can be
carefully withdrawn.
(D) A single sperm cell can be appreciated in the center of the ooplasma
A.single motile sperm is
selectedand immobilized
by pressing its tail
between the microneedle
and the bottom of the dish.
The sperm cell is then
aspirated tail-first into the
injection pipette. B.Using
the holding pipette,
the mature oocyte is fixed
with the polar body at the
6 o0clock position. The
sperm cell is brought to
the tip of the injection
pipette.
ABOUBAKR ELNASHAR

25. 6. Embryo selection
qEmbryos are graded according to:
1. Rate of cleavage: (retarded embryos).
2. Anucleated fragments.
3. Equal sized cells.
§Class A embryos
§Embryos of variable grades.
ABOUBAKR ELNASHAR

26. qScoring for fertilization
§All inseminated or injected oocytes should be
examined for the presence of pronuclei (PN) and
polar bodies at 16 to 18 hours post insemination.
§A normally fertilized oocyte contains 2PN and 2
polar bodies.
§For conventional IVF, cumulus cells must be
removed and 2PN oocytes transferred into new
dishes containing pre-equilibrated culture
medium.
ABOUBAKR ELNASHAR

27. §Abnormal fertilizations.
A : egg with1 pronucleus (PN) B with 3 C with 4 PN.
§Normal fertilization
Oocyte in D, E or F, each has 2 PN.
ABOUBAKR ELNASHAR

28. Fertilization outcome after intracytoplasmic sperm injection.
(A) Oocytes are considered normally fertilized when two
individualized or fragmented polar bodies are present together
with two clearly visible pronuclei (2-PN) that contain nucleoli.
(B) Abnormal fertilization may occur as one pronuclear (1-PN)
oocyte, probably due to parthenogenic activation.
(C) The occasional finding of three pronuclear (3-PN) oocytes
after injection of a single spermatozoon into the ooplasm is
probably caused by non-extrusion of the second polar body at
the time of fertilization.
ABOUBAKR ELNASHAR

29. §Embryos derived from ≥ 3PN oocytes should never
be transferred or cryopreserved.
§Under exceptional circumstances, if no
transferable embryos derived from 2PN oocytes are
available, embryos derived from 1PN oocytes or
oocytes showing no PN but going through normal
cleavage may be used for transfer.
§Even if no transferable embryos derived from 2PN
oocytes are available, the use of embryos derived
from 1PN oocytes or oocytes showing no PN is not
recommended.
ABOUBAKR ELNASHAR

30. The blastomere number is recorded and the embryos are scored accordingly to
equality of size of the blastomeres and the presence of anucleate cytoplasmic
fragments. On day 4 (sometimes already on day 3), a certain degree of
compaction can be observed (E). For blastocyst (F) scoring, the classification
system introduced by Gardner and Schoolcraft is used.
Embryo transfer is usually done on day 3 (eight-cell stage) or day 5 (blastocyst
stage).
Embryo cleavage after
intracytoplasmic sperm
injection. Only embryos
resulting from normally
fertilized oocytes (A) will
be transferred to patients.
Embryo cleavage is
evaluated daily. Two-cell
embryos (B), four-cell
embryos (C), and eightcell
embryos (D) are usually
obtained on day 1 (late
afternoon), on day 2, and
in the morning of day 3,
respectively.
ABOUBAKR ELNASHAR

31. Timeline for optimal blastocyst
development.
ABOUBAKR ELNASHAR

32. 7. Embryo transfer
qEmbryo culture and transfer
§In order to optimise embryo development,
fluctuations of culture conditions should be
minimised.
§Precautions must be taken to maintain adequate
conditions of pH and temperature to protect
embryo homeostasis during culture and handling.
ABOUBAKR ELNASHAR

33. qEmbryo scoring
§should be performed at high magnification (at least
200x, preferably 400x) using an inverted
microscope with Hoffman or equivalent optics.
qEvaluation of cleavage stage embryos should
include
1. cell number
2. size and symmetry
3. percentage of fragmentation
4. Granulation
5. Vacuoles
6. nuclear status (e.g. multinucleation).
ABOUBAKR ELNASHAR

34. qBlastocyst scoring should include
1. expansion grade,
2. blastocoel cavity size
3. morphology of the inner cell mass (ICM)
4. trophectoderm (TE).
qAssessment should be performed at standardised
times post-insemination.
ABOUBAKR ELNASHAR

35. Scoring system for human blastocysts. Initially blastocysts are given a
numerical score from 1 to 6 based upon their degree of expansion and hatching
status: (1) early blastocyst; the blastocoel being less than half the volume of the
embryo; (2) blastocyst; the blastocoel being greater than or equal to half of the
volume of the embryo; (3) full blastocyst; the blastocoel completely fills the
embryo; (4) expanded blastocyst; the blastocoel volume is now larger than that
of the early embryo and the zona is thinning; (5) hatching blastocyst; the
trophectoderm has started to herniate through the zona; (6) hatched blastocyst;
the blastocyst has completely escaped from the zona.ABOUBAKR ELNASHAR

36. qTime-lapse imaging
§Assess embryo development
§more precise evaluation of the timing of
consecutive events
§not interfering with the embryo culture
environment.
ABOUBAKR ELNASHAR

37. qEmbryo selection for transfer is primarily based on
1. developmental stage
2. morphological aspects.
3. Other selection parameters, such as time-
lapse kinetics, may be considered.
ABOUBAKR ELNASHAR

38. qNumber of embryos to be transferred:
§should be based on:
1. embryo quality
2. stage of development
3. female age
4. ovarian response
5. rank of treatment.
§Single embryo transfer is recommended to avoid
multiple pregnancies.
qIt is advisable not to transfer more than two
embryos.
qSupernumerary embryos may be cryopreserved,
donated to research or discarded, according to their
quality, patient wishes and national legislation.
ABOUBAKR ELNASHAR

39. qEmbryo loading:
Air bubbles to bracket the embryo-containing medium
Insufficient evidence to suggest that the fluid-only
method is superior to the use of air brackets during
embryo loading.
(Abou-Setta, 2007)
ABOUBAKR ELNASHAR

40. qTime interval between embryo catheter loading
and discharging the embryos in the uterine cavity
§ The longer the duration, the lower the pregnancy
and implantation rates.
§ The decrease in pregnancy and implantation
rates is gradual until the duration of 120 s, and
decreases sharply afterwards.
(Hum Reprod. 2004)
ABOUBAKR ELNASHAR

41. 9. Cryopreservation
qMethods:
1. Slow freezing
2. Vitrification
ABOUBAKR ELNASHAR

42. qVitrification
§ solidification of a solution (water is rapidly cooled
and formed into a glassy, vitrified state from the
liquid phase) at low temperature
§ Not by ice crystallization but
by extreme elevation in viscosity during cooling.
§ During vitrification the
§ entire solution remains unchanged
§ water does not precipitate: no ice crystals are
formed.
ABOUBAKR ELNASHAR

43. ABOUBAKR ELNASHAR

44. qCryopreservation
Steps:
1.1. Equilibration in the cryoprotectantEquilibration in the cryoprotectant
2.2. Freezing processFreezing process
3.3. Storage in LNStorage in LN22
4.4. Thawing (warming) processThawing (warming) process
5.5. Removal of the cryoprotectantsRemoval of the cryoprotectants
6.6. Culture in the physiological milieuCulture in the physiological milieu
ABOUBAKR ELNASHAR

45. qAction of the cryoprotectant
Penetration of cryoprotectant in the cell andPenetration of cryoprotectant in the cell and
partially replacing thepartially replacing the
↓↓
intracellular waterintracellular water
↓↓
dehydration of the celldehydration of the cell
ABOUBAKR ELNASHAR

46. qCryopreservation can be performed for
Gametes
embryos
tissues.
ABOUBAKR ELNASHAR

47. qSelection of mehod:
according to the type of biological material.
§For sperm:
slow freezing is still the method of choice, but
rapid cooling is a possible alternative.
§For oocytes
vitrification has been reported to be highly
successful and is recommended.
ABOUBAKR ELNASHAR

48. qFor cleavage stage embryos and blastocysts
high success rates have been reported when
using vitrification. However, for cleavage stage
embryos good results can also be obtained
using slow-freezing methods.
qFor tissues
the method of choice is slow freezing, but
vitrification of ovarian tissue is an option.
ABOUBAKR ELNASHAR

49. ABOUBAKR ELNASHAR
You can get this lecture from:
1.My scientific page on Face book:
Aboubakr Elnashar Lectures.
https://www.facebook.com/groups/2277
44884091351/
2.Slide share web site
3.elnashar53@hotmail.com