Male factor itself is responsible for infertility in approx 30-40% couples and contributes to infertility in another approx 20%. In many men having normal sperm parameters on semenogrom, sperms do not function in a manner necessary for fertility and can still cause infertility. This often goes undetected unless specifically sought for. This presentation presents a clinical approach to male factor infertility, specifically related to functional aspects of male fertility.

1. 1
Optimizing male infertility treatment in ART
Dr Parul Katiyar MD
Senior Consultant, Infertility and Reproductive Medicine
Max Hospitals, New Delhi and Gurgaon

2. Male factor infertility
 Male factor is solely responsible for infertility in
~20% cases
 Male factor contributes to infertility in another 30-
40% cases
 Semen analysis is the cornerstone for
diagnosing male infertility
 In many men with normal sperm parameters,
sperms do not function in a manner necessary
for fertility and can still cause infertility

3. How to improve treatment outcome in
ART?
 Improve the diagnostic evaluation
 Improve the sperm health
 Improve the fertilization technique
 Improve the sperm retrieval procedures

4. Improving diagnostic evaluation
 Minimum evaluation of a man with suspected
male infertility includes
 A complete medical history – surgery/ medications/
infections/ allergies/ lifestyle
 Full physical examination – secondary sexual
characters/ testes/ varicocele
 Two semen analyses done eight weeks apart and
assessed using WHO criteria

5. Interpreting semen analysis

6. Further evaluation
 Endocrine 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
 Low/ absent volume ejaculate - < 1ml may be due to
 Ejaculatory duct obstruction
 Retrograde ejaculation
 Anejaculation
 Hypogonadism
 CBAVD
 Scrotal and trans-rectal Ultrasonography  identify and
rule out varicocele, testicular mass, absence of vas and
obstructive pathologies

7. Other investigations
 Sperm DNA integrity
 Genetic screening
 Helps in diagnosing cause of azoospermia
 Helps in counselling 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 microdeletions associated with
isolated sperm impairment

8. Interpreting semen analysis
Focus on Sperm
Health
ICSI/ IMSI/ PICSI
TESA/ TESE/ PESA/
Micro-TESE
Approach to
management

9. Improving health of sperms
 Human sperms play an extensive role that extends
beyond early stages of fertilization to include abnormal
embryogenesis leading to implantation failure and
aneuploidy
 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

10. Why worry about sperm DNA fragmentation?
 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.

11. 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

12. What causes sperm DNA fragmentation?
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.

13. 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
 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

14. Sperm DNA fragmentation assays
 TUNEL assay -> Blue sperm are
TUNEL negative while green sperm
are TUNEL positive indicating DNA
fragmentation.
 Sperm Chromatin Dispersion Test ->
The two sperm in the center with
non-fragmented DNA form large
halos, while the sperm in the upper
right hand corner has no halo
indicating DNA fragmentation.
Results from both assays are expressed as percentage of sperm
demonstrating DNA fragmentation.

15. DNA Fragmentation Index
 Most studies define an upper normal level of the
percentage of cells with DNA fragmentation
 Samples with assay results above this threshold
percentage are considered to have high DNA
fragmentation
 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

16. 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)
 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

17. Sperm DNA fragmentation - Evidence
Authors Study design Outcome
Zini et al (2008) Meta analysis Embryos with high sperm DNA damage are associated with early pregnancy loss
Zini and Sigman
(2009)
Meta analysis Modestly increased pregnancy chance after IVF (OR 1.7, 95% CI 1.3–2.2) in cases
with low level of DNA-damaged sperms
Dumoulin et al
(2010)
In ICSI sperms are injected into the optimal environment of the ooplasm within a
few hours of ejaculation, thus reducing chance of lab-induced damage to sperms
Simon et al (2011) In IVF, couples with low levels of sperm DNA fragmentation (<25%) had LBR of 33%
and the ones with high levels of sperm DNA fragmentation (>50%) had LBR of 13%
Zini (2011) RCT Sperm DNA damage not found to be predictive for ICSI treatment
Aitken et al (2012) Sperms from up to 40% of infertile men have high levels of ROS
Robinson et al
(2012)
High levels of sperm DNA damage are associated with increased risk of pregnancy
loss (OR 2.5, 95% CI 1.5–4.0) regardless of the in-vitro technique applied
Simon et al (2013) OR of 76 (95% CI 8.7–1700) for clinical pregnancy when the mean DNA
fragmentation per spermatozoon was < 52%
Osman et al (2015)Meta analysis LBR was lower for high sperm DNA fragmentation in IVF cycles. But, there was no
difference in LBR between low and high sperm DNA fragmentation with ICSI

18. Laboratory procedures
 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
 Some advanced sperm selection are -
 Sperm selection based on surface charge (Electrophoretic sperm
selection)
 Non apoptotic sperm selection - > Magnetic Activated Cell Sorting
(MACS) System
 Sperm selection by Hyaluronic Acid binding -> PICSI
 Selection based on ultra-morphology -> Motile Sperm Organelle
Morphology Examination (MSOME) -> IMSI

19. Electrophoretic sperm selection
 Positively (PCS) and negatively charged sperms (NCS)
can be identified using micro- electrophoresis
techniques.
 DNA damage is inversely proportional to % NCS and
directly proportional to the % PCS
 Selection of Negatively charged sperms using this
technique helps isolate sperms, which are relatively free
of DNA damage, and can be used for ART
Simon et al. Micro-electrophoresis: a noninvasive method of sperm selection based on membrane charge. Fertility and Sterility, Volume 103, Issue 2,
2015, 361–366.e3

20. Magnetic Activated Cell Sorting (MACS)
System

21. MACS - Evidence

22. 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

23. Tamer M. Said, and Jolande A. Land Hum. Reprod. Update 2011;17:719-733
Sperm selection using PICSI

24. 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

25. 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)

26. MSOME
 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 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

27. IMSI - Grading of Sperms
Grade I – Normal form and no vacuoles
(A)
Grade II – Normal form and ≤ 2 small
vacuoles (B,C)
Grade III – Normal Form, > 2 small
vacuoles or at least one large vacuole
(D,E)
Grade IV – Large vacuole and abnormal
head shapes or other abnormalities (F)
Vanderzwalmen et al. Blastocyst development after sperm selection at high magnification is associated with size and number of nuclear vacuoles.
Reproductive BioMedicine Online 17(5) 617-627

28. Microgragh depicting sperm morphological attributes as assessed by MSOME
(A)Sperm cell with a morphologically normal nucleus
(B)Small oval nuclear form
(C)Large oval nuclear form
(D)Wide nuclear form
(E)Narrow nuclear form
(F)Regional (acrosomal) nuclear shape disorder
(G)Oval nuclear shape and large nuclear vacuoles
(H)Abnormal (narrow) nuclear shape + large nuclear vacuoles
Tamer M. Said, and Jolande A. Land Hum. Reprod. Update 2011;17:719-733
IMSI

29. 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)

30. Role of IMSI - conclusion
 Results from RCTs do not support the clinical use of IMSI
 There is no evidence of effect on live birth or miscarriage
and the evidence that IMSI improves clinical pregnancy is
of very low quality
 There is no indication that IMSI increases congenital
abnormalities
 Further trials are necessary to improve the evidence
quality before recommending IMSI in clinical practice
Teixeira DM, Barbosa MAP, Ferriani RA, Navarro PA, Raine-Fenning N, Nastri CO, Martins WP. Regular (ICSI) versus ultra-high magnification
(IMSI) sperm selection for assisted reproduction. Cochrane Database of Systematic Reviews 2013, Issue 7. Art. No.: CD010167.

31. Improving sperm retrieval procedures
 From the epididymis
 Percutaneous Epididymal Sperm
Aspiration (PESA)
 Microsurgical Epididymal Sperm
Aspiration (MESA)
 From testes
 Testicular Sperm Aspiration (TESA)
 Testicular Sperm Extraction (TESE)
 Microsurgical Testicular Sperm
Extraction (Micro-TESE)

32. 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

33. TESA and PESA
TESA PESA

34. 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

35. Micro-TESE Vs conventional TESE
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

36. Take home messages
Optimizing the outcome of ART in male infertility
Normal semen parameters ≠ healthy sperms
Poor ART outcome can be correlated with high DNA
fragmentation in sperms
Healthy lifestyle & correction of underlying pathology for
healthier sperms
Newer modalities of sperms selection like IMSI, PICSI and
MACS will benefit a carefully selected segment of patients
Micro-TESE is superior to TESE for sperm retrieval in
NOA

37. Thank You
Dr Parul Katiyar
drparulk@gmail.com