Invited Lecture delivered by Dr Sujoy Dasgupta in a Webinar organized by Dr Lal Path Laboratory held in August, 2020

1. Rational Investigations in Male Infertility-
Sujoy Dasgupta
MBBS (Gold Medalist, Hons)
MS (OBGY- Gold Medalist)
DNB (New Delhi)
MRCOG (London)
Advanced ART Course for Clinicians (NUHS, Singapore)
Consultant: Reproductive Medicine, Genome Fertility Centre, Kolkata
Managing Committee Member, Bengal Obstetric & Gynaecological Society (BOGS)
Secretary, Subfertility and Reproductive Endocrinology Committee, BOGS
Convener and Faculty, Spectrum MRCOG Course
Member, Male Infertility, Special Interest Group, ASPIRE, Singapore
Winner, Prof Geoffrey Chamberlain Award, RCOG World Congress, London, 2019

2. CAUSES OF INFERTILITY

3. Global Crisis

4. Esteves S C. Clinical relevance of routine semen analysis and controversies surrounding the 2010
World Health Organization criteria for semen examination. Int Braz J Urol. 2014; 40: 443-53

5. Esteves S C. Clinical relevance of routine semen analysis and controversies surrounding the 2010
World Health Organization criteria for semen examination. Int Braz J Urol. 2014; 40: 443-53

6. WHO Standard 2010
1
2
3
4
5
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7. Murray KS, et al. The effect of the new 2010 World Health Organization criteria for
semen analyses on male infertility. Fertil Steril 2012;98:1428–31
• The 2010 reference values result in some
infertile men being reclassified as fertile if
status is based on semen analysis alone.
• This may lead to fewer men being referred for
proper infertility evaluation or treatment.

8. Limitations of WHO 2010 Semen Analysis
• Based on parameters in a large group of fertile men
along with defined confidence intervals from recent
fathers with known time-to-pregnancy (TTP).
• The WHO does not consider the values set as true
reference values but recommends or suggests
acceptable levels.
• Day to day variation

9. From which Laboratory?

13. Normozoospermia- Further Tests?

14. Routine Semen Analysis- Limitations
• Limitations in predicting the health and
functional capacity of the male reproductive
organs and cells.
• Does not provide information regarding
defects in sperm function.

15. Immunological factors
• Any breach of blood –testis barrier such as trauma/infection/obstruction
• Antisperm antibody present in
1. 10.7% of men undergoing infertility evaluations
2. 10% of men in couples undergoing IVF treatment
3. 42% of men with unexplained infertility
4. only 2% of fertile men
• IgA and IgG
• 27% fertilization rate when >80% sperms had sperm bound ASA
• fertilization rate of 72% when<80% sperms had sperm bound ASA

16. Detection of AntiSperm Antibodies
• Agglutination Tests
• Complement-Dependent Tests
• Immunoglobulin Binding Tests
• Mixed Antiglobulin Reaction and Immunobead Tests
• Enzyme Linked Immunosorbant Assays (ELISA)
• Other tests

17. ELISA
• Antibody-enzyme immunoglobulin
complexes
• add a specific enzyme substrate
→ colour change
• advantage : specific
and quantitative
• disadvantage: the time and cost,
poor sensitivity, and inability to
determine ASA location and
isotype.

18. Sperm DNA Fragmentation
• Advanced paternal age
• Inadequate diet
• Drug abuse
• Tobacco use
• Environmental factors such as pesticide exposure or air
pollution
• Varicocele
• Systemic diseases
• Genital inflammation

19. ESHRE, 2018
• Sperm DNA Fragmentation test is a “Routine”
test in investigating a couple with RPL.

20. What are the lesions associated with Sperm DNA
Fragmentation?

21. SDF and Infertility: Why
bother?

22. IVF outcome and SDF
Meta-analysis of 16 studies
and 2,969 couples
Increased miscarriage in
couples undergoing IVF/ICSI
with high sperm DNA damage
Risk ratio (RR) = 2.16
95% CI: 1.54-3.03; p<0.00001
Robinson et al. Hum Reprod 2012

23. SDF and reproductive success
Points to consider
Gosálbez et al. 2013; Dada et al. 2012
Site of damage
Coding DNA (exons)
represent ~3% of genome

24.  SDF gives different information than routine semen analysis, and of
better prognosticvalue
 SDF is mainly oxidative-stress mediated during sperm transit through
the epididymis
 Elevated SDF associated with infertility, poor ART outcome and
miscarriage
 Reproductive outcome related to oocyte repair capacity as well as
severity and site of DNA damage
SDF and Male Infertility
Key Messages

25. Direct
Incorporation of probes at the site of damage
e.g. TUNEL, ISNT
Indirect
Susceptibility of DBs to denature in a acid solution
e.g. Sperm chromatin structure assay (SCSA), sperm chromatin
dispersion test (SCD), Comet assay
Chromatin compaction
Incorporation of probes to nuclear proteins
e.g. Aniline blue, toluidine blue
Gosálbez et al 2013; Esteves & Agarwal 2011; Esteves et al. 2013
What are the methods for SDF assessment?

26. Enzymatic addition of modified
nucleotides to DNA breaks;
TUNEL (Terminal deoxynucleotidyl transferase
dUTP nick end labeling)
Sharma et al. 2010

27. N
N
TUNEL (Terminal deoxynucleotidyl transferase
dUTP nick end labeling)

28. DNA Fragmentation Index (DFI):
ICSI treatment is more likely to result in pregnancy than IUI and IVF if DFI value is above 30%
Number of TUNEL negative morphologically normal sperm X 100
Total number of sperm cells evaluated
Four statistical types of fertility potency:
Less than or equal to 15% DFI outstanding to sound sperm DNA
credibility
Between 15 to 25% DFI best to good sperm DNA credibility
Between 25 to 50% DFI good to weak sperm DNA credibility
Greater than or equal to 50% DFI exceptionally poor sperm DNA credibility

29. Clinical Management of SDF
• SDF has a negative effect on reproductive potential both in vivo and vitro
• Strategies to reduce SDF include antioxidant therapy, treatment of
subclinical infection, varicocele repair and TESA ICSI
• Avoid iatrogenic damage :short abstinence periods, laboratory sperm
selection and proper sperm handling.
• ICSI is the primary treatment option for patients with a rate of DNA
damage above the established cut off value for the corresponding test
• The effects of spermatozoa with DNA damage being used for fertilization
are still controversial, and further testing is required to assess potential
long-term effects.

30. Pus Cell in Semen

34. What to do
• Empirical Antibiotics?
• Method of collection
• Hand washing before collection
• Special Tests- Round cells vs Pus cells
• History
• Culture of semen
• Prostatic Massage- Culture

35. EUA Guideline, 2018
• The clinical significance of an increased
concentration of leukocytes in the ejaculate is
controversial.
• Although leukocytospermia is a sign of
inflammation, it is not necessarily associated
with bacterial or viral infections.
• More leukocytes found in men with prostatitis
compared to those without inflammation

36. MAGI (Male Accessory Gland Infection)

37. “Mild” Male f=Factor

38. Collection Method Masturbation
Abstinence 4 days
Collection Complete
Volume 2 ml
Colour Whitish
Viscosity Normal
Liquefaction Time 45 minutes
pH 7.6
Sperm Concentration 12 million/ ml
Total Motility 42%
Progressive Motility 17%
Non progressive Motility 25%
Immotile 58%
Motile Sperm Count 20.16 million
Normal Morphology 4%
Vitality 62%
Round cells Nil

39. Collection Method Masturbation
Abstinence 4 days
Collection Complete
Volume 2 ml
Colour Whitish
Viscosity Normal
Liquefaction Time 45 minutes
pH 7.6
Sperm Concentration 12 million/ ml
Total Motility 42%
Progressive Motility 17%
Non progressive Motility 25%
Immotile 58%
Motile Sperm Count 20.16 million
Normal Morphology 4%
Vitality 62%
Round cells Nil

40. Mild Male Factor
• Difficult to define
• Sperm Concentration (SC) >5 million/ ml
• TMSC= Total Motile sperm count = SC x total
volume x TM >5/10/20 million

42. Mild male Factor
• Investigations- NOT usually recommended
• Antioxidants
• CC
• Other adjuvants

43. Oxidative Stress in
Sub-fertility
I n f e r t i l i t y
Oxidative stress (OS) is an imbalance in a cell’s production of
Free radicals( oxidants) of intrinsic or extrinsic origin, and its
ability to reduce them with scavengers.

44. Free Radicals (Oxidants)
Free radical is charged unstable molecule that have at least
one unpaired electron in their outer orbit.

46. Smits RM, Mackenzie-Proctor R, Yazdani A, Stankiewicz MT, Jordan V, Showell MG. Antioxidants for
male subfertility. Cochrane Database Syst Rev. 2019;3(3):CD007411. Published 2019 Mar 14.
• In this review, there is low-quality evidence from seven small
randomised controlled trials suggesting that antioxidant
supplementation in subfertile males may improve live birth rates for
couples attending fertility clinics.
• Low-quality evidence suggests that clinical pregnancy rates may
also increase.
• Overall, there is no evidence of increased risk of miscarriage,
however antioxidants may give more mild gastrointestinal upsets
but the evidence is of very low quality.
• Subfertilte couples should be advised that overall, the current evidence
is inconclusive.

47. When to repeat the semen analysis?
• Mild- After 3 months
• Severe/ Azoospermia- As soon as possible
• (NICE, 2013)

48. Oligospermia and IUI
• TMSC 5-10 mil- Do IUI 4-6 cycles
• TMSC <5 mil- Counsel before IUI
1. Double Ejaculate
2. Post wash- IMSC
3. IMSC >1 mil → Further IUI
4. IMSC <1 mil → see Morphology

49. Tubal Patency before IUI?
• If no risk factors for tubal
block- 3 cycles of IUI,
then tubal patency test
• If risk factors- tubal
patency first
• With severe male factor
chance of tubal factor-
infertility decreases

51. Laboratory testing for Oxidative Stress
Traditional OS laboratory techniques include direct and indirect assessment of OS
Direct Indirect
Chemiluminescence Myeloperoxidase or Endtz test
Nitrobluetetrazolium (NBT) Lipid peroxidation levels
Cytochrome C reduction test Chemokines
Fluoresceinisothiocyanate
(FITC)-labelled lectins
Antioxidants, micronutrients,
vitamins (vitamin E, vitamin C)
Electron spin resonance Antioxidants –TAC
DNA damage

52. External view of the luminometer

53. Setting up of the tubes for ROS measurement by
chemiluminescence assay
The reference value for normal ROS
obtained by this method is <102 relative
light units/s/106 sperm/mL

54. “Severe” Male Factor

55. Collection Method Masturbation
Abstinence 4 days
Collection Complete
Volume 1.5 ml
Colour Whitish
Viscosity Normal
Liquefaction Time 45 minutes
pH 7.6
Sperm Concentration 1.2 million/ ml
Total Motility 30%
Progressive Motility 16%
Non progressive Motility 14%
Immotile 70%
Motile Sperm Count 0.54 million
Normal Morphology 1%
Vitality 34%
Round cells Nil

56. Collection Method Masturbation
Abstinence 4 days
Collection Complete
Volume 1.5 ml
Colour Whitish
Viscosity Normal
Liquefaction Time 45 minutes
pH 7.6
Sperm Concentration 1.2 million/ ml
Total Motility 30%
Progressive Motility 16%
Non progressive Motility 14%
Immotile 70%
Motile Sperm Count 0.54 million
Normal Morphology 1%
Vitality 34%
Round cells Nil

57. Collection Method Masturbation
Abstinence 4 days
Collection Complete
Volume 1.5 ml
Colour Whitish
Viscosity Normal
Liquefaction Time 45 minutes
pH 7.6
Sperm Concentration 1.2 million/ ml
Total Motility 30%
Progressive Motility 16%
Non progressive Motility 14%
Immotile 70%
Motile Sperm Count 0.54 million
Normal Morphology 1%
Vitality 34%
Round cells Nil

58. • Overall, 16 (24.6%) of 65 patients with severe
oligozoospermia developed azoospermia.
• two (3.1%)patients with moderate oligozoospermia
developed azoospermia
• none of the patients with mild oligozoospermia
developed azoospermia.

59. DONOR Sperm is NOT the solution
• Antioxidants- ???
• Consider freezing
• Investigate the cause
• ICSI
• In extreme cases- may need preparation for
TESA

60. • As in azoospermia, in extreme cases of
oligozoospermia (spermatozoa < 1
million/mL), there is an increased incidence
of obstruction of the male genital tract and
genetic abnormalities.

61. Investigation is needed
• If sperm concentration < 5 million/ml

62. History Taking
• Lifestyle
• Medical history- Diabetes, Mumps, Cancer
• Surgical history- Hernia, Orchidopexy, Pituitary
Surgery
• Drugs history- Sulphasalazine, cytotoxic drugs,
steroids
• Sexual history

63. Orchidopexy for B/L Undescended
Testicles

64. Physical Examination
• General body habitus, secondary sex
characters, gynaecomastia
• Testicular size and consistency
• Varicocele
• May diagnose serious disorders

66. Investigations
Endocrine-
If nonobstructive pathology is
suspected
• FSH, LH, Testosterone, sugar
Genetic testing-
1. Karyotyping, Y chromosome
microdeletion- If testicular
failure
2. CFTR testing- If CBAVD
Urological-
• USG Scrotum-
1. Clinically abnormal findings
2. Tight scrotum (Cremasteric
reflex)
3. Post-orchistis (Mumps)
NOT for Varicocele detection
• TRUS-
If obstructive pathology/ ejaculatory
problem suspected

67. Severe OAT/
Azoospermia
Volume/ pH of semen
Normal
Low testicular volume
FSH, LH,
Testosterone, blood
sugar
FSH high
LH Normal/ high
Testosterone normal/low
Karyotyping
YMD
FSH// LH Low
Testosterone low
Pituitary Imaging
FSH/LH Low
Testosterone high
Anabolic steroid
abuse
Prolactin, TSH if
clinically suspected
Volume/ pH of semen low
Normal testicular volume
Scrtotal USG
TRUS
CFTR

68. Severe OAT/
Azoospermia
Volume/ pH of semen
Normal
Low testicular volume
FSH, LH,
Testosterone, blood
sugar
FSH high
LH Normal/ high
Testosterone normal/low
Karyotyping
YMD
FSH// LH Low
Testosterone low
Pituitary Imaging
FSH/LH Low
Testosterone high
Anabolic steroid
abuse
Prolactin, TSH if
clinically suspected
Volume/ pH of semen low
Normal testicular volume
Scrtotal USG
TRUS
CFTR

69. Severe OAT/
Azoospermia
Volume/ pH of semen
Normal
Low testicular volume
FSH, LH,
Testosterone, blood
sugar
FSH high
LH Normal/ high
Testosterone normal/low
Karyotyping
YMD
FSH// LH Low
Testosterone low
Pituitary Imaging
FSH/LH Low
Testosterone high
Anabolic steroid
abuse
Prolactin, TSH if
clinically suspected
Volume/ pH of semen low
Normal testicular volume
Scrtotal USG
TRUS
CFTR

70. Severe OAT/
Azoospermia
Volume/ pH of semen
Normal
Low testicular volume
FSH, LH,
Testosterone, blood
sugar
FSH high
LH Normal/ high
Testosterone normal/low
Karyotyping
YMD
FSH// LH Low
Testosterone low
Pituitary Imaging
FSH/LH Low
Testosterone high
Anabolic steroid
abuse
Prolactin, TSH if
clinically suspected
Volume/ pH of semen low
Normal testicular volume
Scrtotal USG
TRUS
CFTR

71. Severe OAT/
Azoospermia
Volume/ pH of semen
Normal
Low testicular volume
FSH, LH,
Testosterone, blood
sugar
FSH high
LH Normal/ high
Testosterone normal/low
Karyotyping
YMD
FSH// LH Low
Testosterone low
Pituitary Imaging
FSH/LH Low
Testosterone high
Anabolic steroid
abuse
Prolactin, TSH if
clinically suspected
Volume/ pH of semen low
Normal testicular volume
Scrtotal USG
TRUS
CFTR

72. Severe OAT/
Azoospermia
Volume/ pH of semen
Normal
Low testicular volume
FSH, LH,
Testosterone, blood
sugar
FSH high
LH Normal/ high
Testosterone normal/low
Karyotyping
YMD
FSH// LH Low
Testosterone low
Pituitary Imaging
FSH/LH Low
Testosterone high
Anabolic steroid abuse
Prolactin, TSH if
clinically suspected
Volume/ pH of semen low
Fructose Negative
Normal testicular volume
Scrtotal USG
TRUS
CFTR

73. In this case
• FSH 15.21 IU/L (normal 1-10)
• LH 12.8 IU/L (normal 1-10)
• Testosterone 159 ng/dl (normal 200-800 )

74. Testosterone Supplementation?

75. Estrogen in male?
• Estradiol , normal range- 10-40 pg/ml
• If T:E2 ratio <10 (T- ng/dl, E2- pg/ml),
consider Aromatase Inhibitors
(Anastrozole 1 mg/day or letrozole 2.5 mg/day)

76. Asian J Andr, 2019. A systematic review and meta-analysis of clinical trials
implementing aromatase inhibitors to treat male infertility

77. Ultrasound of Scrotum
• Should NOT be done just to detect subclinical
varicocele

78. Case-Study
• 34-yrs-old, Army-man,
primary infertility
• Repeated examination
showed total
asthenospermia
• Past smoker
• Ejaculated sperms-
poor morphology
• Advocated for TESA-
ICSI done, conceived

79. Varicocele
• Subclinical: not palpable or visible, but can be
shown by special tests (Doppler ultrasound
studies).
• Grade 1: palpable during Valsava manoeuvre, but
not otherwise.
• Grade 2: palpable at rest, but not visible.
• Grade 3: visible at rest.

80. Surgery in varicocele

81. Case-Study
• 32-yrs-old IT
Professional
• Already received
antioxidants
• Uncontrolled diabetes
was diagnosed after
finding OAT
• 5 cm (large)
epididymal cyst/
Spermatocele
• IUI was planned
• Natural conception

82. While the occurrence of epididymal cysts in this cohort is unexplained, our observation
that these cysts are not associated with infertility will be useful for those clinicians
counseling patients observed to have these structures.

83. Mumps-Orhitis, Secondary Azoospermia

84. Self testicular Examination
• Atrophic Testes
• H/O undescended testicles
• Testicular microcalcification

85. Obstructive Azoospermia

86. Collection Method Masturbation
Abstinence 2 days
Collection Complete
Volume 0.5 ml
Colour Whitish
Viscosity Normal
Liquefaction Time 45 minutes
pH 6.8
Sperm Concentration Nil
Round cells Nil

87. Collection Method Masturbation
Abstinence 2 days
Collection Complete
Volume 0.5 ml
Colour Whitish
Viscosity Normal
Liquefaction Time 45 minutes
pH 6.8
Sperm Concentration Nil
Round cells Nil

88. Assess
• Abstinence period
• Completeness of collection
• Usual amount of ejaculate
• Exclude retrograde ejaculation
• Post-masturbation urine
• Suspect obstructive pathology- TRUS

89. Surgical Sperm Retrieval in
Azoospermia (OA>NOA)

90. Case Study
• 38-yr, IT
worker
• B/L vas not
palpable
clinically

91. CFTR testing was done after trial TESA
confirmed presence of motile sperms

92. Clinical features related with infertility
male: atrophy, fibrose or congenital absence of vas deferens
female: reduced fertility, thick dehydrated mucus in the cervix
CFTR mutations- CAVD
About 98% of males affected with CF are infertile
Congenital Absence of Vas Deferens (CAVD)
1-2% male infertility, 6% obstructive azoospermia
Mutations (>1300) in CFTR gene (Cystic Fibrosis
Transmembrane Conductance Regulator)

93. CFTR mutations -CAVD
The deltaF508 deletion is the
most common cause of cystic fibrosis.
The isoleucine (Ile) at amino acid
position 507 remains unchanged
because both ATC and ATT code for
isoleucine

94. Non-obstructive Azoospermia

95. Collection Method Masturbation
Abstinence 5 days
Collection Complete
Volume 3.0 ml
Colour Whitish
Viscosity Normal
Liquefaction Time 45 minutes
pH 7.8
Sperm Concentration Nil
Round cells Nil

96. Collection Method Masturbation
Abstinence 2 days
Collection Complete
Volume 3.0 ml
Colour Whitish
Viscosity Normal
Liquefaction Time 45 minutes
pH 7.8
Sperm Concentration Nil
Round cells Nil

97. Predictors of sperm retrieval?
• FSH
• Testicular Size
• No reliable positive prognostic factors
guarantee sperm recovery for patients
with non-obstructive azoospermia.
• The only negative prognostic factor is
the presence of AZFa and AZFb
microdeletions.

98. Trial TESA
• No role of FNAC
• If possible, freeze the sperms

99. Case-Example 1

100. Karyotyping

101. Klinefelter syndrome-Karyotype 47,XXY

102. Chromosome Microarray Analysis

103. Role of Microarray
Can detect any sub-microscopic CNVs like Y-chromosome microdeletion

104. Role of Microarray

105. Case-Example 2

106. Y Chromosome Microdeletion
• Deletions are too small to be detected by karyotyping

107. Microdeletions in Yq11 (AZFa, AZFb, AZFc) are the most
frequent genetic cause of male infertility after KS

108. AZFc Deletion

109. Y-Chromosome microdeletion PCR
• AZFa: sY81, sY84s, sY86, sY182
• AZFb: sY121, sY133, sY124,
sY127, sY128, sY130, sY134,
sYPR3
• AZFc: sY157, sY254, sY255,
sY145, sY152, sY242, sY208
• SRY: sY14
Y chromosome deletion analysis. The amplification products from Multiplex A,
B,C,D and E master mixes are shown. The sample DNA (Lane S) depicts deletions
(highlighted in yellow) when compared to Male Genomic DNA control (Lane C).
The marker (M) lanes contain the 50bp DNA Step Ladder.

110. Case-Example 3

111. YMD Report

112. Patient refuses donor sperm in
presence of genetic defect
• Sperm Aneuploidy testing by FISH
• PGT-SR (previously- PGD)

114. Sperm Aneuploidy Detection by FISH
Limitation
This technique allows the detection of aneuploidy for the limited number of
chromosomes included in the test. In very few ejaculated samples or testicular
samples, is there not enough spermatozoa for a proper estimation of the risk of
aneuploidy.

115. Wyrobek et al., 1990
Four-chromosome FISH for detecting aneuploid human sperm
Sperm Aneuploidy Detection by FISH

116. Sperm Aneuploidy FISH Panel at LPL
Normal sperms

117. Sperm Aneuploidy FISH Panel at LPL
Abnormal sperms

118. Ejaculatory Disturbance

119. Case-Study
• 38-yrs-old, teacher, married for 5 years
• Unable to ejaculate during coitus and also during
masturbation since marriage
• Nocturnal emission present
• Diabetic on medication for last 8 years
• Initially used to ejaculate during masturbation
• Physical exam unremarkable
• Vibroejaculator failed
• Post-masturbation urine- No sperms

120. TRUS
• TESA-ICSI done,
conceived, delivered

121. Ejaculation Problems
• DM
• Spinal cord Injury
• Neurological diseases
• Obstruction
• Medication- SSRI, PDE-5 blockers, alpha-
blockers
• Exclude- retrograde ejaculation

123. Take Home Messages
• Semen analysis must be done from reliable
laboratories, following WHO 2010 standards
• Single abnormal test must be repeated
• Antioxidants should be offered in mild male
factor problems
• In severe problem, active investigation and
treatment should not be delayed
• Donor-Sperm is NOT the only solution