Sperm Factors on Embryo
Implantation Potential
Engin Enginsu M.D. Ph.D.
Kadıköy Şifa Hospital, ART Unit
Spermed Laboratory
2008
Semen Analysis
Minimum 2 sperm analysis
Ejaculate Volume
Sperm Count
Total Motility
Progressive Motility
Normal Morphology (MEUSC)
Additionally
Agglutination
Piospermia
Viscosity
1.5 to 5.0 mL
>20 million/mL
>30%
>30%
>4%
None
None
Normal
Makler Counting Chamber
Semen Analysis
Phase contrast attachment
20 x 10 magnification
Morphology
Head
Smooth oval shape
Acrosome 40-70%
Equatorial segment
Mid-piece
Tail
10x100 magnification
Results of Semen Analysis
Astheno, 2
Oligo, 9
Normal, 50
Terato, 10
Azo, 6
OAT, 7
OT, 11
OA, 3
AT, 2
n=2610
SPERMED Laboratory 2008
Azoospermia
Centrifuge
Suspend pellet with 0,3 ml culture
medium and vortex
All pellet should be evaluated on slide
Effects of Sperm Anomalies
Decrease in Sperm-ZP
binding
Oligozoospermia, severe
teratozoospermia
ZP-induced acrosome
reaction
Oligozoospermia
ZP penetration problems
Teratozoospermia
(Liu et al. 2004)
Acrosome Reaction
Acrosome is a membranebound cap-like structure
covering the anterior part
of the sperm head,
containing:
Acrosine
Neuroaminidase
Hyaluronidase
Antibodies or
Fluorescent probe
Vitality Test
Globozoospermia
Globozoospermia
Syndrome characterized by spherical
chromatin structure and absence of acrosome
DNA structure unknown
Increase in sperm aneuploidy rate (case
report)
Abnormal chromatin structure, DNA strand
breaks
Diagnosed by morphological evaluation
(Calogero et al. 1999 – 2001-2002)
Globozoospermia
(Dam et al. 2007)
Globozoospermia
Responsible genes and inheritance is unknown
Heterologous ICSI: Centrosomal function defect,
decrease in oocyte activation
Decrease in fertilization and oocyte activation
Pregnancy with ICSI, decreased fertilization
rate
Calcium ionophore enhances fertilization rate
(Dam et al. 2007, Rubes et al. 1998, Nakamura et al. 2002)
Globozoospermia
No spontaneous pregnancy
Aneuploidy rate increases in sex
chromosomes and 13, 14, 15, 16, 18,
21
(Dam et al. 2007, Ditzel et al. 2005)
Macrosephalic Sperm
Macrosephalic Sperm and PGD
(Kahraman et al. 2004)
Macrosephalic Sperm
70
60
46.7
50
40
14.3
Abortion
Rate
Imp. Rate
30
25
20
10
12.3
0
Before PGD
After PGD
(Kahraman et al. 2004)
DNA Damage
DNA integrity is crucial for fertilization
Occurs in testicular, epididymal and postejaculatory levels
DNA strand breaks can cause chromosomal packaging
abnormalities
High DNA damage ratio
Embryonic development arrests
Apoptosis
Implantation abnormalities
Early pregnancy loss
(Evenson et al., 1999; Morris et al., 2002)
DNA Damage
DNA strand breaks are related with
Age
Schizophrenia
Achondroplasia
Apert's syndrome
Sperm count
Sperm motility
Blastocyst culture can be a good alternative to
detect paternal inheritance in ART
DNA Damage
(Lewis et al., 2005)
DNA Damage
Comet Assay (Single Cell Gel Electrophoresis Assay)
Sperm cell electrophoresis on agarose gel
Evaluation of DNA migration
Acridine Orange
Same principals with SCSA – Evaluation of
results are difficult
Halosperm
Detection of DNA breaks - FISH
Halo evaluation after denaturation with acid
and removal of nuclear proteins
Comet Assay
ABNORMAL
NORMAL
Sperm Chromatin Structure
Assay (SCSA)
Acridine Orange Test,
Aniline Blue
Fluorescent microscopy
Flow cytometry
Green normal, red
DNA breaks
DNA Breaks = Red/Red +
Green
More specific than Comet
Assay and TUNEL Test
Green Fluorescence
DNA Damage
COM
Pat
Red Fluorescence
at
Advanced technology
required
(Lewis et al. 2008)
Halosperm (Sperm Dispertion Test)
Fragmanted
Sperm heads with
different sizes
DNA intact sperms
sorted by size
Fragmanted DNA
shows no halo in
nuclear region
Normal
Enciso et al, 2006
TUNEL Test
Detection of single and double strand
breaks in DNA
TdT-mediated dUTP nick end labeling
(TUNEL)
Most common method for DNA break
evaluation
Fast and easy
Lewis et al, 2008
TUNEL: Terminal deoxynucleotidyl transferase
mediated dUTP-biotin Nick End Labelling
NORMAL CHROMATIN
APOPTOTIC CHROMATIN
TESE: Alternative Approach
TESE + ICSI in high DNA breaks
Invasive
TUNEL: Reliability?
Sensivity and specificity???
Cut-off value???
Centrosome?
(Greco et al., 2005)
Sperm Centriole Defects
Sperm centriole enters oocyte with fertilization,
duplicates and forms sperm aster
Centriole abnormalities
Embryonic developmental defects
Implantation failures
Preclinical abortion
(Van Blerkom, 1996 Rawe et al., 2002
Sperm Centriole Defects
Dithiothreitol (DTT) and Taxol restores
sperm centriole function in Dysplasia of
the fibrous sheath (DFS)
Reliability? (Heterologue ICSI/bovinehuman)
(Nakamura et al., 2005)
Sperm Centriole Defects
Post fertilization embryo development
disorder or block
Sperm centriole in the sperm tail
attachment site can not be transferred
to active zygote centriole
(Rawe et al., 2008)
Aneuploidy in Sperm
Sperm Chromosome Anomaly Ratio in
patients with Normal Somatic Karyotype
FERTILE
OLIGOZOOSPERMIA
TERATOZOOSPERMIA
˜ %0,3 - 1,08
˜ %0,7 - 9,44
˜ %1,3 - 3,90
Aneuploidy in Sperm
Although ICSI is a solution for male
infertility, it causes inheritance of genetic
abnormalities
Non-obstructive Azoospermia
Severe OAT
Obstructive Azoospermia
Ejaculatory Sperm
11.4-24.9 %
18 %
1.8-5.8 %
1.5-2.3 %
Aneuploidy in Sperm
Anomaly ratio in chromosomes X, Y and 18;
Sperm with Fragmented DNA v.s. Normal
Sperm
4.6 fold anomaly
Diploidy and disomy is 4.4 and 5.9 fold,
respectively
Anomaly ratio in chromosomes X, Y and 17
1.5-4 times more in immature than mature
sperms
(Muriel et al., 2007)
Aneuploidy in Sperm
The frequency of aneuploidies, especially
disomies, is significantly lower in sperm from the
80% Percoll fraction, that is, enriched in mature
spermatozoa compared to the unprocessed semen
sample.
Immature sperms have more fragmented DNA
(Muriel et al., 2007)
Sperm Sex Chromosome Disorders
Normal morphology
6
% Non-Disjunction
5
4
3
2
1
0
18
X
Y
24XX
Infertile / Oligozoospermia
Increase in the number of sex chromosomes in sperm
Non-obstructive Azoospermia
Sex chromosome disorders in ~40 % of germ cells
Inheritance risk in most of the anomalies
Testicular Sperm
Sperm maturation
Sperm motility
Frozen sperm
Testicular Sperm
Fresh and frozen testicular sperm
Fertilization
Pregnancy
Implantation
Live Birth
Fresh
%79.3
%39.6
%25.4
%19.5
Frozen
%71.1
%25
%8.0
%6.0
N.S.
N.S.
<0.05
<0.05
De Croo et.al. 1998
Testicular Sperm
Motility in testicular samples
Motility
Fertilization
Implantation
Clinical pregnancy
r-FSH stim.
%68.8
%20.1
%47.9
Control
%42.1
%13.2
%30
Balaban et.al. 1999
Testicular Sperm
Increase in DNA damage in sperms, which
are incubated or frozen, in Obsturictive
Azoospermia patients
Incubation of testicular sperm is only
beneficial for motility and morphology
(Lewis et al., 2005, Dalzell et al. 2004)
DNA Damage in Testicular Sperm
High Magnification ICSI
Hazout a. 2006, RBM Online
High Magnification ICSI
Hazout a. 2006, RBM Online
Conclusions
Semen analysis reveals important
information on genital and testicular
function.
Fertilization disorders can be detected
with sperm function tests.
Sperm DNA damage is related with
reproductive disorders, however,
insufficient for explaining all pregnancy
losses.
Conclusions
Aneuploidy screening can be an alternative
in abnormal semen analysis results.
Time dependant DNA damage can occur in
testicular sperm. Thus, timing of ICSI
after TESE should be shortened.
ICSI is not sufficient for sperm function
disorders.
Conclusions
More studies needed for sperm centriole
defect treatments.
Blastocyst culture is the most current
treatment for paternal disorder
eradication