Sperm DNA Fragmentation

Sperm DNA Fragmentation -
Causes & Guidelines for Current Treatment

Dr. Sasikala Natarajamani MBBS,MMEDSCI(ART) EMB(ACE

www.creaconceptions.com

Flow
1. Human Sperm Cell
2. Structure of Human Sperm Chromatin
3. Causes of Sperm DNA Damage
4. Type of DNA Damage
5. Effect on Reproductive Outcomes
6. Tests for Diagnosis
7. Usefulness of the Tests
8. Guidelines for Current Practice


The Sperm Cell
• Sperm cell different from other cells in the body
• Small size –at the expense of cytoplasm –cell mass
• Reduced Cell mass
– Impaired production of enzymes required for genetic
repair
• Chromatin in somatic cells –
– Relatively loose structure
• Chromatin in sperm because of small size –
– Very tightly compacted- haploid genome must adapt
to a volume 40 times less than a somatic cell


Sperm Chromatin Structure

• Fundamental packaging unit of sperm chromatin
is a toroid which has 50-60 kb of DNA
• Individual Toroids represent dna loop-domains
highly condensed by protamines fixed at nuclear
matrix
• Toroids are cross linked and further compacted
by di - sulphide bonds
• Each chromosome is a garland of Toroids


• During later stages of Spermatogenesis – spermatid nucleus remodelled and
condensed

• Histones displaced by transition proteins and then by Protamines which are
more basic and much smaller than histones (Steger et al, 2000).

• Unique associations between the DNA and Protamines.

• DNA strands are tightly wrapped around the Protamine molecules forming
tight and highly organized loops (Brewer et al, 1999).

• Intermolecular and intra-molecular disulphide cross-links between Protamines
lead to compaction and stabilization of the sperm nucleus

• Nuclear compaction - protect the sperm genome from external stresses
(Kosower et al, 1992).


• Sperm nuclear proteins are predominantly Protamines,
to a lesser extent histones- about 15%

• In many species – Protamines form 95% of nuclear
proteins but in humans – only 85% is Protamine bound
and 15% DNA is packaged by histones at specific DNA
sequences

• Histone bound DNA are less tightly compacted

• When the strands are not packed well - long DNA
strands susceptible to damage – Sperm DNA
fragmentation www.creaconceptions.com


Source : http://molehr.oxfordjournals.org/content/16/1/30/F1.expansion

Causes of Sperm DNA Damage

• Intrinsic factors
– Remodeling and Packaging Problems
– Protamine Deficiency
– Excess ROS
• Damage by ROS
– Abortive Apoptosis

• Extrinsic factors
– Chemotherapy
– Cigarette smoking
– Genital tract inflammation
– Testicular hyperthermia
– Varicoceles

Intrinsic Factors


Remodelling & Packaging Problems

• Stage-specific transient DNA strand breaks are
introduced during Spermiogenesis

• DNA breaks are needed for transient relief of torsional
stress, favouring the histone replacement with
protamines during the final maturation from round to
elongated spermatozoa

• These physiological, temporary breaks if not repaired –
lead to DNA fragmentation or genetic mutations in the
ejaculate (Marcan and Boissoneault 2004)


Protamine Deficiency

• Infertile men – ↑ histone to protamine ratio ( Cho et al
2001, Aoki et al 2005,2006)
• Complete lack of protamines in some ( zhang et al
2006)
• Deficiency → defective chromatin compaction → ↑
susceptibility
• Transgenic animal models with targeted protamine
deficiency – show link between protamine deficiency,
DNA damage, fertilizing capacity at IVF
– (Cho et al 2001, 2003)
• Probably linked to defective Spermiogenesis – when
protamines are deposited ( Steger et al 2000)

Excess ROS levels
• ROS – Important Physiological role – Modulate Gene &
Protein activities vital for Sperm Proliferation

• Physiological amounts are controlled by seminal antioxidants
– imbalance between ROS production and ability of
antioxidants to scavenge

• Excess ROS levels - pathological ( Zini et al 1993, Irvine et al
2000)

• Excess – generated by morphologically defective sperms
(residual cytoplasm in particular) and semen leukocytes →
DNA damage


Damage by ROS

• Spermatozoa are prone to damage by free oxygen radicals
due to the abundance of poly unsaturated fatty acids which
are essential for fertilization

• Free oxygen radicals activate caspases and endonucleases
which are also induced by physiochemical factors including
heat exposure – leading to DNA fragmentation


Causes - Oxidative Stress


Abortive Apoptosis

• Apoptosis of testicular germ cells occurs throughout life

• Prevents over proliferation and selectively aborts
abnormal forms (Sinha Hikin and Swerdolff 1999)

• Some sperms have initiated but escaped apoptosis -
abortive apoptosis ( Sakka et al 2003)


Extrinsic Factors


Extrinsic factors
• Radiotherapy, chemotherapy and environmental toxins
–induce sperm DNA damage

• Can be direct effect on dna or indirect effect by
changing the endocrine milieu of the testis or epididymis

• Leads to lowered activity of the testosterone-dependent
DNA enzyme topoisomerase

• Reduced production of antioxidants by epididymis


2 Step Hypothesis
Faulty spermatogenesis→ defective remodeling → DNA
more susceptible to stress factors

Aitken & De Iullius 2010


Type of DNA damage
• Both single stranded breaks as well as double stranded
breaks are possible
• SSB is easy to repair and better prognosis
• SSB – due to unrepaired DNA nicks and ROS
• DSB – abortive apoptosis, action of caspases and
endonucleases & ROS
• DSB – unrepaired – gross alteration to chromosomal
structure
• DSB – more serious and deleterious impact on
development of progeny


Effect on Reproductive Outcome

• Oocytes and early embryos have been shown to repair
sperm DNA damage.
• the biological effect of abnormal sperm chromatin
structure – is the combined effect of sperm chromatin
damage and the capacity of the oocyte to repair the
damage
• Fertilization is independent of dna damage
• Post fertilization development is affected by improper
repair by the oocyte → implantation failure, early
miscarriages, diseases in the offspring


Diagnostic Tests
• Comet –single cell gel electrophoresis
• Tunnel –terminal deoxy nucleotide transferase mediated
UTP nick end-labeling
• Sperm chromatin dispersion
• Sperm chromatin structure assay


Diagnostic tests

• All these tests label single or double stranded DNA breaks
• Comet & Tunel – detect actual DNA strand breaks – measure
existing damage
• Others measure the susceptibility of DNA to denaturation –
formation of single stranded DNA from native double stranded DNA
– hence includes potential future damage
• SCSA– flow cytometry test - measures the susceptibility of DNA to
acid induced denaturation ( Evanson and Jost 1994, Evanson et al .,
2002)
• So far only SCSA is the only test with clear and clinically useful cut
off levels for calculating male fertility potential


Diagnostic tests
• Most studies define an upper normal level of the percentage of cells
with DNA fragmentation.

• Unit of measurement – DFI ( DNA fragmentation Index)

• Samples with assay results above this threshold percentage are
considered to have high DNA fragmentation.

• Percentage of spermatozoa with fragmented DNA
≤ 15 – good fertility potential
15-25% - average
> 25% - poor fertility potential


Usefulness of Tests -Disadvantages

• Most assays do not differentiate between clinically significant and
insignificant dna damage
• Some DNA nicking occurs as a normal process during winding and
unwinding of DNA; current assays do not differentiate physiologic
from pathologic nicking.
• Assays do not evaluate the genes that may be affected by the
fragmentation
• Fragmentation in areas containing certain genes may be more
detrimental than fragmentation in relatively inactive regions of the
genome.
• Assays simply determine the amount of DNA fragmentation - based
on the belief that more DNA nicking or fragmentation is pathologic.


Usefulness of Tests
• No conclusive evidence - studies give conclusions
ranging from “very useful” to “ not useful” or “more
research is needed”
• Confusion partly due to lack of standardization of the
methodology for dna testing
• Low number of patients for studies
• Majority of studies on ART do not report on the methods
used to achieve a pregnancy( IUI, IVF ICSI)


Usefulness of tests
• Lack of consistency in findings of different investigators – uncertain
significance of high DFI Speyer et al.,2010

• ASRM practice committee 2006: tests of DNA damage are
insufficiently predictive of pregnancy outcome

• Meta analysis of Evanson and Wixon 2006 – High DFI has less
effect on ICSI than IVF

• Meta analysis of Lei et al.,2006 – Tunel assay- high DFI showed
significant effect on outcome of IVF not ICSI
• SCSA – no effect on IVF of ICSI


What are the relevant questions?

• Do the DNA damage tests predict the chances of
achieving a spontaneous pregnancy?

• Do they help to choose the appropriate method of ART?

• Are these tests immune to intra and individual variation?


DNA integrity & Spontaneous Pregnancy

• Very few studies

• Spontaneous pregnancy chances reduce when DFI exceeds 30%

• In half of the cases of “unexplained infertility” sperm DNA damage is
a contributing factor

• In patients with oligo astheno terato zoospermia
chance of increased chromatin damage is high


DNA Testing & Selection of ART

• In couples with DFI >27% no pregnancy can be achieved
regardless of type of ART
Larson-Cook et al., 2003

• 2004 – 3 independent studies –DFI >27% -compatible
with pregnancy and delivery
- ART compensates for poor chromatin quality
Virro et al.,2004,Gandini et al., 2004, Bungum et al., 2004


Variations in Tests
• Normal S/A – inter and intra individual variations

• Similarly even in DFI- such variations exist - Keel et al.,
2006

• Biological background – not clearly known but lifestyle
factors play a major role

• Individuals with specific genetic polymorphisms may be
more susceptible to these environmental pollutants


Current Practice - Guidelines
• Unexplained infertility , normal semen parameters with mild or
treatable female infertility
• DFI > 25-30% - direct IVF/ICSI
• DFI < 30% - treatment of female- spontaneous pregnancy can be
tried.
– A. Giwercman et al .,2011
• Minor impairment of semen parameters
• Concentration, Motility, Morphology - below reference range and
short period of infertility
• DFI <10% -female <35 years, no pathology – spontaneous
pregnancy can be tried
• DFI>10% - treatable female subfertility – treatment of the female


Current practice -Guidelines

• Couples referred for IUI with standard semen
parameters

• DFI > 25-30% - direct IVF/ICSI

• DFI> 30% - ICSI considered


Current Practice -Guidelines

Giwercman et al 2011


Role of DNA Damage Tests

• Role in clinical practice is still widely debated
• Proven role in reproductive and environmental research
• Majority studies –small patient cohorts
• Most studies focus on difference between fertile and infertile patients
in percentage of sperms with DNA damage
• Do not focus on predictive value of these tests
• Powerful indicator of in vivo fertility
• Between IVF and ICSI more related to IVF than ICSI
• Clinical threshold values established only for SCSA – methodology
highly standardized ( low inter and intra laboratory variation)


Conclusions
• Successful human reproduction depends on the inherent integrity of the
sperm DNA.

• There appears to be a threshold of sperm DNA damage beyond which
embryo development and subsequent pregnancy outcome are impaired.

• Clinical evidence shows that, sperm DNA damage is detrimental to some
reproductive outcomes and that the spermatozoa of infertile men possess
substantially more DNA damage than that of fertile men.

• Our understanding of the etiology of sperm DNA damage, and the full
impact of this sperm defect on reproductive outcomes in humans, still
remains rudimentary

• Therapies for impaired DNA integrity need to be developed and subsequent
improvements in sperm DNA integrity from such therapies need to be
correlated with improved reproductive outcomes
.

Thank You


Sperm DNA Fragmentation

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