2. Over 300 ppts are available on slideshare.net
***for use of public/Doctors
www.slideshare.net / Lifecarecentre
3. SEMEN ANALYSIS
No doubt it Provides useful
information concerning sperm
production by the testis, sperm
motility and viability, the
patency of the male genital
tract, the secretions of the
accessory organs, as well as
ejaculation and emission
4. NORMAL VALUES OF SEMEN VARIABLES:
WHO GUIDELINES
1999 2010
Volume 2.0 mL or more 1.5 ml (1.4–1.7);
pH 7.2 to 8.0
Sperm concentration 20 million or more 15 million per ml (12–16);
Total sperm count : 40 million or more 39 million per ejaculate (33–46);
Motility : 50% or more with forward
progression or 25% or more with
rapid progression
progressive motility, 32% (31–34);
total (progressive + non-
progressive) motility, 40% (38–42
Morphology 30% or more with normal forms 4.0% (3.0–4.0).
White blood cells Less than 1 million
Immunobead MAR<50% bound/adherent
4
6. • Conventional semen analysis
limited for MALE fertility
assessment; caution should be
applied when interpreting
results
7. INTERPRETING SEMEN ANALYSIS
Normal semen
parameters
• Despite looking
normal, sperms
may still be
damaged
• RO species
damage DNA
fragmentation
Poor semen
parameters
• Oligospermia
• Asthenospermia
• Teratozoospermia
Azoospermia
• Obstructive
• Non obstructive
8. BIG QUESTION
HOW TO IMPROVE
PREGNANCY RATES IN
IUI ,IVF,& ICSI if
semen parameters
are Normal???
9. INTERPRETING SEMEN ANALYSIS
Normal semen
parameters
• Despite looking
normal, sperms
may still be
damaged
• RO species
damage DNA
fragmentation
Poor semen
parameters
• Oligospermia
• Asthenospermia
• Teratozoospermia
Azoospermia
• Obstructive
• Non obstructive
Focus on Sperm
Health
ICSI/ IMSI/ PICSI
TESA/ TESE/
PESA/
Micro-TESE
Approach to
management
10. 4 KEY Areas
To IMPROVE OUTCOME IN ART?
• Improve the diagnostic evaluation
• Improve the sperm health
• Improve the fertilization technique
• Improve the sperm retrieval procedures
11. HARMONAL evaluation is
indicated when there is
–An abnormally low sperm count
(<10 m/ ml)
–Impaired sexual functions
–Early findings suggestive of
endocrinopathy – base evaluation
includes S FSH and Testosterone
FURTHER DIAGNOSTIC
EVALUATION1
12. Clinical Diagnosis based on
Hormonal Status
Clinical Status FSH LH Testosterone
Normal
men or obstruction Normal Normal Normal
Isolated
spermatogenic failure Normal Normal
Testicular failure
Normal or
Hypogonadotropic
hypogonadism
13. • Genetic screening
– Helps in diagnosing cause of azoospermia
– Helps in counseling the would be parents about risk of
transmission to offspring
• 3 main genetic factors known to be related to male
infertility are:
– Cystic fibrosis gene mutation
– Chromosomal abnormalities resulting in impaired testicular
functions
– Y chromosomal micro deletions associated with isolated
sperm impairment
GENETIC SCREENING
15. • Scrotal and trans-rectal
Ultrasonography identify and
rule out varicocele, testicular
mass, absence of vas and
obstructive pathologies
IMAGING
In Male Infertility
17. SPERM DNA FRAGMENTATION
Our experience shows that Sperm DNA
Fragmentation
is valuable laboratory tool
for clinical decision making
As
Conventional semen analysis not
enough…should be offered early in
workup.
18. Most common cause of poor sperm
health is DNA fragmentation
DNA fragmentation can be
• Single strand DNA break
• Double strand DNA break
• Base deletion or modifications
• Inter and intra-strand cross linkage
19. SPERM DNA DAMAGE IS A
USEFUL BIOMARKER FOR
Male infertility diagnosis
(better marker than conventional semen analysis)
prediction of assisted reproduction outcomes
• SPERM DNA FRAGMENTATION IS ASSOCIATED WITH
– Reduced fertilization rates
– Poorer embryo quality
– Poorer pregnancy rates
– Higher rates of spontaneous miscarriage
– Higher incidence of childhood diseases.
Schulte RT, Ohl DA, Sigman M, Smith GD. Sperm DNA damage in male
infertility: etiologies, assays, and outcomes. Journal of Assisted
Reproduction and Genetics. 2010;27(1):3-12.
20. WHO BENEFITS FROM SPERM
DNA ASSAY?
• Couples with unexplained infertility
• Couples with history of unsuccessful ART
• Couples with history of miscarriages
• Men over 30 years of age
• Men at higher risk of OS
– Men with diabetes
– Men with history of drug abuse
– Men who have been treated for cancer
21. WHAT CAUSES SPERM DNA
FRAGMENTATION?
Intrinsic factors
• Protamination failure
• Oxidative stress during
transit or after
ejaculation
• Apoptosis during
sperm maturation
inside the tubules
Extrinsic factors
• Life style – Smoking/
Obesity
• Varicocele
• GT Infections
• Radiation exposure
• Exposure to toxins
such as Lead
Denny Sakkas and Juan G. Alvarez. Sperm DNA fragmentation:
mechanisms of origin, impact on reproductive outcome, and
analysis. Fertility and Sterility Vol. 93, No. 4, Pages 1027-36.
22. METHODS TO DIAGNOSE DNA
FRAGMENTATION
• Comet – single cell gel electrophoresis
– Detects actual DNA strand breaks and measures existing
damage
• Tunnel –terminal deoxy nucleotide transferase
mediated UTP nick end-labeling
– Detects actual DNA strand breaks and measures existing
damage
23. METHODS TO DIAGNOSE DNA
FRAGMENTATION
• Sperm chromatin dispersion (SCD) -> “halosperm” test
(normal sperms have a halo)
– Measures the susceptibility of DNA to denaturation –> formation of
single stranded DNA from native double stranded DNA
– Estimates potential future damage
• Sperm chromatin structure assay (SCSA)
– Measures the susceptibility of DNA to acid induced denaturation
– Estimates potential future damage
– SCSA is the only test with clear and clinically useful cut off levels for
calculating male fertility potential
24. SPERM DNA FRAGMENTATION ASSAYS
Results from both assays are expressed as percentage of sperm demonstrating
DNA fragmentation.
25. DNA FRAGMENTATION INDEX
• INTERPRETATION OF VALUES
– Spermatozoa with fragmented DNA ≤ 15% –> good fertility
potential
– Spermatozoa with fragmented DNA 15-25% -> average fertility
potential
– Spermatozoa with fragmented DNA > 25% -> poor fertility
potential
26. 80% of couples with diagnosis of
IDIOPATHIC infertility have sperm DNA
damage
27. Effects of High Sperm DNA
Fragmentation
DNA Fragmentation Levels are Closely
correlated with
IUI,
IVF and
ICSI
outcomes
and pregnancy rates
29. • Live birth rates with IUI
• as a function of SDF
1.5%
Normal Elevated
Normal
N=387; OR = 0.07 !
[95% CI: 0.01-0.48]
Bungum et al. Hum Reprod 2007 !
IUI outcome impacted by sperm chromatin
integrity
30. Meta-analysis of 16 studies;
2,969 couples:
Increased miscarriage in IVF/
ICSI cycles when high SDF:
RR = 2.16
95% CI: 1.54-3.03; p<0.00001
Robinson et al. Hum Reprod 2012
Pregnancy rate in cases of
elevated sperm DNA
fragmentation
Bungum et al. Hum Reprod 2007 !
26%
42%
IVF ICSI
IVF outcome impacted by sperm chromatin
integrity
31. MANAGEMENT OF SPERM DNA
FRAGMENTATION
• LIFESTYLE MODIFICATIONS
– Cessation of smoking and alcohol intake
– Weight management for obese men
• Antioxidants – should be used at least for 2-3 months
for effect
– Vit C (500 mg/ day)
– Vit E (200 mg/ day)
– Folic Acid (2 mg/ day)
– Zinc (25 mg/ day)
– Selenium (26 mcg/ day)
32. MANAGEMENT OF SPERM DNA
FRAGMENTATION
• Treatment of underlying pathological conditions
• Varicocele
– GU Infections
• Avoid environmental exposure to toxins and judicial medical
use of radiations
• Reducing the abstinence period or serial ejaculation
every 24 hours reduces SDF by up to 25%
• In cases with uncorrected DNA fragmentation – consider
surgical sperm retrieval
35. Varicocele
• Patient with varicocele have higher
proportion of sperm with massive
DNA damage
• Varicocele surgical repair
significantly reduce SDF
(Wang et al. RBM online 2012)
38. GREAT TIP
• Microsurgical varicocele repair
and use of TESTICULAR SPERM
for ICSI may improve ART
outcomes in selected individuals
39. MACS
Magnetic Activated Cell Sorting
• Magnetic separation of healthy
spermatozoa by maintaining the structure,
viability & functionality of the sperm.
• Aimed to be used in IUI,
IVF-ICSI cycles as the sperm
can be used in real time.
41. • ICSI – motility & morphology
• Invisible anomalies ignored
• Novel techniques implemented to ensure
selected sperm have intact chromatin
• DNA damage represented by fragmentation &
subsequent apoptosis- male infertility
MACS
Magnetic Activated Cell Sorting
42. DNA fragmentation due to apoptosis can be
inherent or can be due to external factors.
MACS helps to remove such apoptotic sperm –
supramagnetic beads conjugated to antibody to
Annexin V.
MACS
Magnetic Activated Cell Sorting
45. ANTIOXIDANTS
• use of Antioxidants and Good diet
has almost become universal to
optimize pregnancy outcome
2
46. LIBERAL USE OF
ANTIOXIDANTS
Protein rich diet,
Vitamins
micronutrients &
antioxidants to be
liberally started
2 – 3 months
prior to actual IVF
47. Vitamin – C 500 mg O – D daily
Vitamin – E 400 mg B – D daily
Are most important supplementation
******
Lyco – Q 300 mg daily is the 3rd option if
they can afford
ANTI-OXIDANT IN MALE
49. PICSI
• ICSI remains the gold standard embryology procedure to tackle
Male Infertility
• No major breakthrough after ICSI to tackle male infertility and
improve outcome of ART
• Current target of research is to further improve outcome of
treatment is to find techniques which will identify sperms
with best fertilization potential…PICSI is very useful in
our experience
50. Tamer M. Said, and Jolande A. Land Hum. Reprod. Update 2011;17:719-733
Sperm selection using PICSI
51. SPERM SELECTION BY
HA BINDING - PICSI
• Formation of hyaluronic acid binding sites on the
sperm plasma membrane is one of the signs of sperm
maturity and forms the basis of sperm selection.
• PICSI dish has been developed by adding 4 marked
spots of immobilized HA in a Falcon petri dish.
• One drop of washed spermatozoa is placed at the edge
of HA spot and HA bound spermatozoa are collected
after 15 minutes in an ICSI pipette and used for
injection
52. ADVANTAGES & EFFECT
ON ART OUTCOME
Advantages of PICSI
• PICSI ensures that the selected sperms are mature ->
Defined by Creatinine Kinase, HspA2 & Aniline Blue staining
• Lower risk of aneuploidy
• Better embryo quality and cleavage rates.
Effects on ART outcome – what does the evidence tell
us?
• Not much improvement in fertilization rates/ pregnancy
rates.
53. HA BINDING/ PICSI - EVIDENCE
Author (year) Outcomes reported
Ye et al (2006)
HBA score when FR >50 versus HBA score when FR ≤50%:
75% versus 69% (marginal S)
Nasr-Esfahani et al
(2008)
FR 79% versus 68% (S)
PR 46% versus 40% (NS)
Tarozzi et al (2009)
FR when HBA score ≥80% versus FR when HBA score <80%:
86% versus 87% (NS)
PR when HBA score ≥80% versus PR when HBA score <80%:
36% versus 32% (NS)
Van Den Bergh et al
(2009)
FR 76% versus 70% (NS)
PR NA
Parmegiani et al (2010)
FR 92 versus 86% (NS)
PR 25% versus 21% (NS)
Parmegiani et al (2010) FR 93% versus 87% (NS)
54. • Sperm morphology is a major
determinant of male in vivo and
in vitro fertility
• Sperms selection method has been developed
based on the inclusion of only normal sperms
assessed using motile sperm organelle
morphology examination at a magnification of
6300X
MSOME
55. MSOME
MSOME assesses six sperm organelles in real time
– Acrosome -> categorized as normal or abnormal
– Post-acrosomal lamina -> categorized as normal or
abnormal
– Neck -> categorized as normal or abnormal
– Tail -> categorized as normal or abnormal
– Mitochondria -> categorized as normal or abnormal
• Nucleus for shape and chromatin content
(vacuolar areas) -> most important part of
assessment
56. IMSI
• ICSI - performed under an inverted
microscope with magnification of 200×-400×,
that allows to detect both motility and
normal morphology of spermatozoa, based
on evaluation of their head, neck and tail.
• this selection has certain limitations –
– low magnification used that permits to observe
only major sperm morphological defects
57. • A new method of human spermatozoa
evaluation called “motile sperm organelle
morphology examination” (MSOME)
• MSOME allows spermatozoa examination
at high magnification (6000× and over)
– a better morphological evaluation
– the selection of motile spermatozoa with a morphologically
normal nucleus, normal nuclear
– content and mitochondrial function. The integration of MSOME
method into ICSI procedure led to intracytoplasmic
morphologically selected sperm injection (IMSI)
IMSI
58. IMSI - EVIDENCE
Authors Findings of study
Bartoov et al (2002)
Normal nucleus by MSOME when pregnant versus not pregnant 34%
versus 25% (S)
Bartoov et al (2003) FR 63% versus. 64% (NS); PR 66% versus 30% (S)
Berkovitz et al (2005) FR 71% versus 50% (S); PR 52% versus18% (S)
Berkovitz et al (2006) FR 73% versus 69% (NS); PR 50% versus 18% (S)
Berkovitz et al (2006) FR 67% versus 69% (NS); PR 48% versus 20% (S)
Hazout et al (2006) FR NA; PR 38% versus 2% (S)
Antinori et al (2008) FR NA; PR 39% versus 27% (S)
Mauri et al (2010) FR 71% versus 70% (NS)
Gianaroli et al (2008) FR 74% versus 72% (NS); PR 31% versus 21% (NS)
Gianaroli et al (2010) FR 69% versus 67% (NS); PR 55% versus 14% (S)
59. EVIDENCE ON IMSI
• A meta-analysis of randomized controlled
trials (RCTs) concluded that the
current evidence does not support
the clinical use of IMSI
since there is no effect on live birth or
miscarriage rate, and that the quality of the
scientific evidence for a beneficial effect on the
clinical pregnancy rate is of very low quality.
62. SPERM RETRIEVAL TECHNIQUES-
INDICATIONS
Technique Indications Success Rate
PESA • OA cases only
MESA • OA cases only
TESA
• Failed PESA in OA
• CAVD
• Favorable testicular pathology in NOA
• Previously successful PESA in NOA
15 -50%
TESE
• Failed PESA or TESA in OA
• NOA cases 20 - 60%
Micro - TESE • NOA cases only 40 - 67%
Esteves SC et al. Sperm Retrieval Techniques for Assisted Reproduction. Int Braz J Urol 2011
Esteves & Agarwal. Sperm Retrieval Techniques. Cambridge University Press, 2011
64. WHAT IS MICRO-TESE
• Method to identify the site(s) of production - based on the
diameter of seminiferous tubules
• Microsurgical approach
– Identify the site of production
– Preserve the vasculature of testis
– Excise small quantity of testicular tissue
Testicular sperm extraction: microdissection improves sperm yield with minimal
tissue excision. Peter N. Schlegel. Hum.Reprod. (1999) 14 (1): 131-135. Hum.
Reprod. (1999) 14 (1): 131-135.
An isolated region of morphologically normal
spermatogenic tubules seen within an
otherwise poorly functioning testis
65. Micro-TESE Vs conventional
TESE
45%
93%
64%
20%
25%
64%
9%
6%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Overall Hypospermatogenesis Maturation Arrest Sertoli-cell only
Sperm Retrieval Success Rate
Micro-TESE Single biopsy TESE
p = 0.02
p < 0.01
Prospective controlled study on 60 patients)
Verza & Esteves, Microsurgical versus conventional single-biopsy testicular sperm
extraction in nonobstructive azoospermia: a prospective controlled study, Fertility
and Sterility, Volume 96, Issue 3, Supplement, September 2011, Page S53
Micro-TESE more effective than conventional TESE
66. TAKE HOME MESSAGES
Optimizing the outcome of ART in male infertility
• Normal semen parameters does not mean healthy sperms
always
• Poor ART outcome can be correlated with high DNA
fragmentation in sperms
•
67. Optimizing the outcome of ART in
male infertility
•Healthy lifestyle & correction of underlying
pathology for
healthier sperms
• Newer modalities of sperms selection like
PICSI and
MACS will benefit a carefully selected
segment of patients
• Micro-TESE is superior to TESE for sperm
retrieval in NOA
68. ADDRESS
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Karkari Morh Flyover,
Delhi - 51
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Editor's Notes
Sperm selection using PICSI dishes. (A) A sperm drop is placed at the periphery of a HA drop, mature sperm binds to the HA-spot, while immature sperm moves freely. (B) Bound sperm could be picked up with the ICSI pipette. (Jakab et al., 2005, with permission from Elsevier.).