Role of sperm on

embryo quality
Dicky Moch Rizal
Dept. of Physiology, Fac.of
Medicine, GMU
 Pregnancy

– Fertilization

– Implantation :
 Embryoquality
 Endometrium/site of implantation
 Embryo quality

– Sperm quality

– Egg quality
 Semen Analysis:
World Health Organization Guidelines

Parameters Normal
range Volume
1.5 - 5 mL
Sperm conc. >20
million/mL
Sperm motility >50%
Sperm morphology >30%
normal forms
Leukocyte density <1
million/mL

 Need at least 2 S/As

 SPERMPARAMETER NOT ENOUGH TO
PREDICT FOR SPERM FUNCTION !!

 Neednew marker for measuring

male fertility potential
 Thecompletion of fertilization
process and subsequent of embryo
development need sperm DNA
integrity

------------ succesfull or failure !!

 Sperm DNA damage :
– DNA fragmentation
– Abnormal packaging of chromatin
– Protamine deficiency
 Human sperm chromatin structure is
very important for producing the
best sperm quality

 Predominantlywith protamine (85%)

and histone more less (15%)

 Increasing
of histone lead to poor
sperm quality
 Why protamine..?
– During late spermatogenesis..important
phase of spermatogenesis
– The DNA strands are tightly bound/
wrapped around the protamine
molecules
– Responsible for nuclear compactions
– Important to protect from oxidation
stress and high temperature exposure
 essential
for sperm head
condensation and DNAstabilization
 In humans and maybe other
primates, 10-15% of the sperm's
genome is packaged by histones
thought to bind genes that are
essential for early embryonic
development
 the chief protein components
of chromatin, acting as spools
around which DNA winds, and play a
role in gene regulation
 Epigenetic (heritable changes in gene expression or cellular
phenotype caused by mechanisms other than changes in the
underlying DNA sequence mechanisms)

– (e.g. gene potentiation, interactions

with the nuclear matrix,histone
acetylation, DNA methylation) are at
play in the regulation of this group of
genes, and how the impact of these
factors may change throughout
differentiation to mature spermatozoa
 Reactive Oxygen Species (ROS)
and Male Infertility
25% infertile men have
high semen ROS levels
n +
Semen ROS
Infertile 166
25%
Fertile 12
0

Semen ROS levels

correlate negatively
 Biology of ROS
ROS are ubiquitous in aerobic
biologic systems

Physiologic: diverse cellular functions

and cellular differentiation

Pathologic: cellular aging (enzyme

inactivation, DNA breaks, lipid
peroxidation), tumorogenesis
 Biology of ROS
Pathology stems from imbalance
between production and scavenging
Production
Degradation
 ROS and ROS Reactivity
t½ Strength
Potential Targets

.O - seconds +
2
SHgps, NADH
H2O2 >> seconds ++
DNA, SHgps
.OH << seconds ++++
Lipids,DNA,SHgps
NO. seconds ++
 Source of High ROS in Semen

Leukocytes
(WBCs)

Spermatozoa
 Source of ROS in Semen: WBC
WBC concentration
correlates with ROS
levels in whole semen
“Outlying” points
represent samples
with significant
sperm-derived ROS
production

Aitken & West, Int J

Androl, 1990
Kovalski et al, Fertil Steril,
1991
 Source of ROS in Semen:
Spermatozoa
The generation of
ROS by human
spermatozoa occurs
spontaneously under
aerobic conditions

Defective sperm
function (e.g.
motility) is
associated with the
accumulation of lipid
peroxides

MacLeod, Am J Physiol,
1943
 Source of ROS in Semen:
Spermatozoa

Retained cytoplasm
(RC)
– midpiece area
correlates with ROS
production
– midpiece area
correlates with
sperm dysfuntion
(motility,
fertilization in vitro)

Gomez et al, J Androl, 1996

Keating et al, J Reprod
 ROS and Sperm Dysfunction
Mechanism of ROS-induced sperm
dysfunction:
– Peroxidation of sperm membrane
lipids (accumulation of lipid
peroxides)
– ATP depletion
– Oxidation of proteins/SH-groups
– DNA oxidation/fragmentation
Alvarez et al, J Androl, 1987
Aitken et al, Biol Reprod, 1989
de Lamirande & Gagnon, J Androl, 1992
Mammoto et al, Biol Reprod, 1996
 ROS and Sperm Dysfunction:
Fertility in Vivo
High levels of semen ROS are
associated with low pregnancy rates in
vivo
Aitken et al, Am J Obst Gynecol, 1991
No ROS

Intermedia
te

High
ROS
 ROS and Sperm Dysfunction
Mechanism of ROS-induced sperm
dysfunction:
– Peroxidation of sperm membrane
lipids (accumulation of lipid
peroxides)
– ATP depletion
– Oxidation of proteins/SH-groups
– DNA oxidation/fragmentation
Alvarez et al, J Androl, 1987
Aitken et al, Biol Reprod, 1989
de Lamirande & Gagnon, J Androl, 1992
Mammoto et al, Biol Reprod, 1996
 Successful IVF/ICSI with Damaged
Sperm DNA: Implications
DNA oxidation may
cause errors in DNA
replication and repair
after successful
fertilization
Kuchino et al, Nature,
1987

Potential for de
novo Genetic
Human Reproduction, Vol. 18, No. 5, 1023-1028, May 2003
Sperm DNA fragmentation – embryo quality
based on morphology criteria
 Drugs thought to induce male
infertility
– Anti-androgens : Spironolactone, cimetidine, flutamide
– Androgen suppressors :Ketoconazole & leuprolide
– Oestrogens & hormones : Oestrogen agonists, growth
hormone, & anabolic steroids
– Drugs of abuse : Anabolic steroids, alcohol, marijuana,
cocaine & nicotine.
– Psychoactive agents : Tricyclic antidepressants,
amphetamine, tranquillisers & phenytoin.
– CVS agents : Propanolol, digoxin & Ca2+ channel
antagonists.
– GIT & antibiotics :.Sulphasalazine & nitrofurantoin
Role of Antioxidants in Semen
Function
– Protect normal sperm from ROS-
producing sperm
– Protect normal sperm from WBC-
derived ROS
– Suppress premature sperm
maturation
Site of Action
– Male reproductive tract
– Female reproductive tract
 Antioxidant Activity in Semen
Enzymatic & Non-Enzymatic
Antioxidants
– SOD
– Catalase
– Glutathione peroxidase
– Taurine / Hypotaurine
– Vitamin C / E
– Urate
– Lycopene
Alvarez et al, J Androl, 1987
Alvarez & Storey, Gamete Res, 1989
Jeulin et al, Gamete Res, 1989
Holmes et al, J Androl, 1992
Zini et al, Int J Androl, 1993
Palan & Naz Arch Androl 1996
ROS and Normal Sperm Function

Low levels of oxidative stress in vitro

enhance:
– sperm hyperactivation
– sperm capacitation
– acrosome reaction
– sperm-egg binding
– sperm-egg fusion
– Fertilization
Bize et al, Biol Reprod, 1991
de Lamirande et al, Int J Androl, 1993
Griveau et al, Int J Androl, 1994, 1995
Zini et al, J Androl, 1995
Kodama et al, J Androl, 1996
Aitken et al, Biol Reprod, 1998
Role of Antioxidants in Semen
Function
– Protect normal sperm from ROS-
producing sperm
– Protect normal sperm from WBC-
derived ROS
– Suppress premature sperm
maturation
Site of Action
– Male reproductive tract
– Female reproductive tract
 Etiology of Oxidative Stress:
ROS Production or Antioxidant
Deficiency?

Production
Degradation
 Etiology of Oxidative Stress:
ROS Production or Antioxidant
Deficiency?
 Primary oxidant excess?
WB
Studies C
Aitken et al, Hum Reprod, 1995
Gomez et al, J Androl, 1996
RO
S
 Primary antioxidant deficiency?
Studies
Zini et al, Int J Androl, 1993
Smith et al, Hum Reprod, 1996
Lewis et al, Fertil Steril, 1995
Sanocka et al, J Androl, 1996
Alkan et al, J Urol, 1997
 Treatment of
Oxidative Stress-Mediated
Infertility
1. Disease-Specific Therapy
Antibiotic therapy: for genital tract infection
Varicocelectomy: to improving sperm
morphology and decrease the % of
spermatozoa with retained cytoplasm
Vasquez-Levin et al, J Urol, 1997
Zini et al, Hum Reprod, 1999
Lifestyle Changes: to reduce oxidants (e.g.
cigarette) Therapy (in vitro)
2. Antioxidant
Beneficial Effect of Antioxidants (in
vitro) on Sperm Motility
Antioxidants may be
effective in
preserving sperm
motility in aerobic AOX1
conditions
– Albumin
– Taurine
– Hypotaurine

CTL AOX2,3
Alvarez & Storey, Biol
Reprod, 1983
Twigg et al, Hum Reprod,
1998
 Treatment of
Oxidative Stress-Mediated
Infertility
1. Disease-Specific Therapy
Antibiotic therapy: for genital tract infection
Varicocelectomy: to improving sperm
morphology and decrease the % of
spermatozoa with retained cytoplasm
Vasquez-Levin et al, J Urol, 1997
Zini et al, Hum Reprod, 1999
Lifestyle Changes: to reduce oxidants (e.g.
cigarette)

2. Antioxidant Therapy (in vitro)

3. Antioxidant Therapy (oral)

 Oral Antioxidant Therapy:
Vitamins C & E
Study Agent Rx Placebo Outcome Comment

Fraga, ‘91 Vit C 28 --- DNA oxidatn No placebo

Dawson, ‘92 Vit C 50 25 spz quality ?abstinence
Kessopoulou,‘95 Vit E 15 15 egg-binding otherwise neg
Suleiman, ‘96 Vit E 52 35 motil, pregn Drop-out (25%)
Rolf, ‘99 Vit C/E 15 16 negative small

*
 Oral Antioxidant Therapy:
Zinc, Folic Acid, Selenium

Study Agent Rx Placebo Outcome

Wong,‘02 Zn +/- FA 75 25 spz quality with combined Rx

Scott,‘98 Se 23 23 spz motility

Vezina,’96 Se + Vit E 9 --- spz motility, morphology

*
 Lycopene and Male Infertility:
Overview
1. Male Infertility
2. Oxidative Stress and Male
Infertility
3. Antioxidant Therapy for Male
Infertility
4. Lycopene and Male Infertility
 The most important cellular
contribution of the sperm cell to the
zygote is the centrosome. During
embryo development the sperm
centrosome forms the poles of the
mitotic spindle, thereby regulating
the first and subsequent cell
divisions
 It has been ascertained that the
blastocyst formation rate as well as
blastocyst morphology were
significantly lower when sperm with
impaired quality were used in
fertilisation of oocytes in
conventional IVF procedures
 theintegrated head density (IHD) is
able to measure DNA breaks that are
irreparable in the long term, leading
to failure in the later stages of
embryonic development
 Assisted Conception

 IUI: intrauterine insemination

 IVF: in vitro fertilization

 ICSI: intracytoplasmic sperm

injection
 GIFT: gamete intrafallopian transfer

 ZIFT: zygote intrafallopian transfer

 PESA: percutaneous epididymal

sperm aspiration
 Hum Reprod. 2008 Dec;23(12):2663-8. Epub 2008 Aug 29.

 Sperm DNA damage is associated

with an increased risk of pregnancy
loss after IVF and ICSI: systematic
review and meta-analysis.
 Zini A, Boman JM, Belzile E, Ciampi A.

 sperm DNA damage is associated with a significantly increased risk of

pregnancy loss after IVF and ICSI.
 J Assist Reprod Genet. 2011 May;28(5):391-7. Epub 2011 Feb 16.

 Is sperm dna damage associated

with IVF embryo quality? A
systematic review.
 Zini A, Jamal W, Cowan L, Al-Hathal N.

 there is no consistent relationship between sperm DNA damage and

embryo quality and/or development. The data also suggest that the
influence of sperm DNA damage on embryo quality/development may be
more significant in ICSI compared to IVF cycles.
 .
Syst Biol Reprod Med. 2011 Feb;57(1-2):78-85. Epub 2011 Jan 6

 Are sperm chromatin and DNA

defects relevant in the clinic?
 Zini A.
 A systematic review of the literature allows us to conclude that sperm DNA
damage is associated with lower natural, intra-uterine insemination (IUI),
and in vitro fertilization (IVF) pregnancy rates. Studies to date have not
shown a clear association between sperm DNA and chromatin defects and
pregnancy outcomes after intra-cytoplasmic sperm injection (ICSI)
 However, we cannot exclude the possibility that very high levels of DNA
damage will impact on ICSI outcomes. In couples undergoing IVF or ICSI,
there is evidence to show that sperm DNA damage is associated with an
increased risk of pregnancy loss