ICSI IVF

1. Intracytoplasmic Sperm Injection
Factors affecting fertilization and division of Embryos
By
Wael Abdelrahman Alheleily
Embryologist
B. Sc. (Chemistry/ Botany). Fac. Sci. Benha Univ. 2007
Analytical biochemistry Diploma, Fac. Sci. Menoufia Univ. 2009
Premaster in applied microbiology, Fac. Sci. Benha Univ. 2010
M.Sc. In genetic engineering, (Genetic Toxicology), (GEBRI)

2.  The ICSI has become method of choice to achieve fertilization
 Injection is possible with sperm obtained from ejaculation, microsurgical epididymal sperm
aspiration (MESA), percutaneous epididymal sperm aspiration (PESA), or testicular sperm
extraction (TESE).
 Total failed fertilization (TFF) refers to failure of fertilization in all mature oocytes and
“failed fertilization” refers to failure of fertilization in any mature oocyte.
 TFF occurs in 5–10% of IVF (MahutteN. G.AriciA.2003 ) and 1-3% of ICSI cycles
(FlahertyS. P.PayneD.MatthewsC. D.1998) TFF after ICSI cycles is mostly due to low
number of mature oocytes
• Some patients may face repeated TFF in spite of normal sperm parameters and good
ovarian response (TesarikJ.RienziL.UbaldiF.et al.2002). In such cases, the primary reason
for failed fertilization after ICSI is lack of oocyte activation,
1. Introduction

3. 2- Factors Affecting Fertilization
A- Sperm B- Oocyte

4. A - Sperm related factors
Sperm structural defects
• Normal sperm ultrastructure correlates with positive ICSI results.
• Sperm morphology is unique among the known cells and 3 major parts can be immediately
distinguished: head, Midpiece and tail.

5. Abnormal sperm morphology is classified as defects
in the head, Midpiece or tail of the sperm.
 A sperm head vacuole is considered abnormal when it exceeds 20% of the head's cross-
sectional area.
 There is a strong correlation between high relative vacuole area to sperm head and
poor sperm morphology.
 Macrocephalic and large-headed spermatozoa are commonly associated with a low
chance of pregnancy, mainly in relation to meiotic abnormalities during spermatogenesis.
Head defects
 Globozoospermia results from perturbed expression of nuclear proteins or from an
altered Golgi-nuclear recognition during spermatogenesis.

6.  Fertilization after ICSI using round-head sperm is inability of sperm to
activate the oocyte.
Globozoospermia

7. Midpiece defect
 Include 'bent' neck (where the neck and tail form an angle of greater than
90% to the long axis of the head), asymmetrical insertion of the midpiece into
the head, a thick or irregular midpiece, an abnormally thin midpiece, as well
as any combination of these.

8. Tail defects
Include short, multiple, hairpin, broken or bent (>90°) tails, tails of irregular width, coiled tails, as well as any
combination of these.

9. Sperm DNA damage
 Concentration, motility and morphology are parameters commonly used to
determine the fertilization potential of an ejaculate.
 Sperm DNA fragmentation (SDF) tests can also differentiate fertile from infertile
males and that high levels of SDF are positively correlated with lower fertilization
rates in ICSI, impaired implantation rates and an increased incidence of abortion .
Protamine
 Replace the majority of histones
 As the sperm pass through the epididymis, the protamines are cross-
linked by disulphide bonds reducing the chromatin to one-sixth the
volume taken up in somatic cell nuclei
 This dense compaction gives protection against exogenous assault to
the sperm DNA .

10. Tests are currently available to evaluate SDF
Sperm Chromatin Structure Assay (SCSA)
the TUNELAssay
the In Situ Nick Translation (ISNT)
the DNA Breakage Detection(DBD)
Fluorescence in Situ Hybridization(FISH)
the Comet Assay
the Single-cell pulsed-field gel electrophoresis technique
Sperm Chromatin Dispersion Test (SCDt)

11. Sperm Chromatin Dispersion Test (SCDt)

12. Semen processing for Ejaculate sample
Count and Motility sample

13.  DNA damage in the male germ line is associated with poor fertilization
rates
 Many published articles indicate that DNA strand breaks are clearly
detectable in ejaculated sperm and their presence is heightened in the
ejaculates of men with poor semen parameters.
 Testicular samples show a significantly lower DNA damage compared
to ejaculated spermatozoa (14.9%±5.0 vs. 40.6%±14.8, P<0.05),
(MoskovtsevS. 2012).
 No correlation is observed between DNA defect and sperm-head
morphology.

14. What about Immotile sperms ??!!
 In case of immotile sperm, it is possible that the sperm may be dead.
The most common practice to select viable non-motile sperm for ICSI
involves the hypo-osmotic swelling (HOS) test.
 Upon exposure of the sperm to hypo-osmotic conditions, the
intact semi-permeable barrier formed by the sperm membrane
allows an influx of water and results in swelling of the cytoplasmic
space and curling of the sperm tail fibers.
 Obtaining viable spermatozoa from testicular biopsies using
pentoxifylline is more effective in terms of fertilization and pregnancies
than obtaining it through an HOS test.

15. Hypo-osmotic swelling (HOS) test

16. Azoospermia
Obstructive Non-obstructive
Result of obstruction in either the
upper or lower male reproductive tract.
Sperm production may be normal but
the obstruction prevents the sperm
from being ejaculated.
Result of testicular failure where sperm
production is either severely impaired or
nonexistent, although in many cases sperm
may be found and surgically extracted directly
from the testicles.

17. Stages of Oocyte maturation (Normal morphology)
B – Oocyte
A and B are GV, C is MI and D is MII oocyte

18. Abnormal morphology
Oocytes in both rows show extra-cytoplasmic and cytoplasmic
dysmorphism.
Oocytes represent different degrees of vacuoles in cytoplasm. Each oocytes
in second row has increased central granularity.

19. Proper orientation of the polar body and needle position
 Breaking the tail is thought to damage the cell membrane and invoke
subsequent physiological and biochemical reactions that may promote
decondensation of the sperm head and activation of the oocyte (Parrington et al,
1996).
 Injection of a motile sperm without immobilization leads to poor fertilization rates.
 Cytoplasmic maturity is thought to be asynchronous with nuclear maturity in stimulated
cycles and the fertilizing ability of an oocyte with a mature nucleus is not necessarily at
its maximum potential. Thus, preincubation of oocytes prior to IVF or ICSI may induce
cytoplasmic maturation that could eventually increase fertilization and also pregnancy
rates.

20.  The embryologist performing ICSI procedure is a significant predictor of fertilization, and
laboratory conditions (i.e. incubators, culture of oocytes individually versus grouped) do not
affect the rates.
 When fertilization failure in most or all of the injected oocytes occurs, with experienced
practitioners using normal sperm, the diagnosis falls to oocyte dysfunction, oocyte activation
failure, or inability of sperm to be decondensed and processed by the oocyte.

21.  The preincubation period between oocyte retrieval and injection improves the
percentage of mature oocytes, the fertilization rate, and the embryo quality.
E shows typical funnel that appears after ICSI, F shows leakage of ooplasm after
ICSI, G shows oocyte damage during denudation and H is an atretic oocyte after
ICSI

22. .
A is an egg with1 pronucleus (PN), B with 3 and C with 4 PN. All these are
abnormal fertilizations. Second row: Oocyte in D, E or F, each has 2 PN.
This is a sign of normal fertilization.
Normal and abnormal fertilization after ICSI

23.  The zona-free oocytes may be fertilized normally after ICSI and develop to
the blastocyst stage (JelinkovaL.et al.2001), Pregnancy in human and live
birth in mouse have been obtained after transfer of embryos resulting from
zona-free oocytes.
zona-free oocytes Development of embryo without zona

24.  Significant advances have been made in achieving fertilization, pregnancy and
live birth in cases with severe male factor infertility, oocyte activation failure and
ICSI technique. Usually fertilization is 80-100 percent in mature eggs, however,
low or no fertilization can still occur. Most cases of no fertilization occur due to
very low number of mature oocytes, failure of oocyte activation or non-
availability of appropriate sperm. Repeated ICSI attempts results in fertilization
in 85% of cases.
Conclusion
 Repeated ICSI treatment can be useful or necessary because there is a high
possibility of achieving normal fertilization if a reasonable number of oocytes with
normal morphology are available and motile sperm can be found. If there are no
motile sperm present in the first ejaculate, a second sample should be required
followed by PESA or TESE to obtain motile sperm. In this way, a sufficient number of
motile sperm for ICSI are usually found in most men with severe
asthenozoospermia.