Male Infertility is a inability that causes pregnancy in a female fertile. Male infertility is commonly due to Low sperm Count. Soi provides best male infertility treatment in delhi, ghaziabad - India. For more information call us 9810350512

1. NEWER MODALITIES
FOR SEMEN TESTING
DR GAURI AGARWAL
FERILITY SPECIALIST(DIRECTOR IVF)
SEEDS OF INNOCENCE IVF CENTRE-
COLLABORATED WITH UNIVERSITY OF
GENT,BELGIUM

2. Introduction
Primary infertility affects  15% of couples
Male infertility accounts for 50 % of cases
Evaluation of Male partner includes :
– Detailed history
– Physical examination,
– Semen analyses,
– Genetic & hormonal evaluation,
and
– Specialized semen tests

4. SEMEN ANALYSIS

5. Standard reference values for semen
characteristics (WHO 2010)
Parameter Lower Reference Limit
Semen volume (ml) 1.5
Sperm concentration (106/ml) 15
Total sperm number (106/ejaculate) 39
Progressive motility (PR, %) 32
Total progressive motility (PR +NP, %) 40
Vitality (live sperms, %) 58
Sperm morphology (NF, %) 4
pH* >/=7.2
Leucocyte* (106/ml) <1
MAR/Immunobead test* (%) <50

7. FUNCTIONAL TESTS

8. SPERM VITALITY TESTS

9. • Vitality assessment done with:
1. Eosin-nigrosin (dead sperm stain pink/red)
2. Eosin (1%) (dead sperm stain pink/red)
3. Trypan (0.4%) blue (dead sperm stain blue)
4. Hypo-osmotic swelling test (HOS) (live sperm shows
tail curling
• Test 1, 2 and 3 for diagnostic uses
– Usually 1:1 ratio of semen to dye mixture, mixed well &
smear onto a slide
– Read immediately at x40 objective, count 200 sperms
Vitality Assessment

10. • Test 4 is use to choose live (immotile) sperm for
Intracytoplasmic sperm injection (ICSI)
– Dead sperms will not react in HOS
– While live sperm will take up fluids causing their tails to
curl within 5 min and stabilize at 30 min.
– Therefore viable sperms may be selected for ICSI
Vitality Assessment

13. Vitality Assessment
Dead sperms are stained pink/red Live sperms shows curling tails

15. CASA

17. • Uses video & computer software technology to capture types &
speed of sperm motility.
• Automatic image digitization & processing
• Additional parameters can be measured such as curvilinear
velocity (VCL) , straightline velocity (VSL), linearity and flagellar
beat frequency and amplitude of lateral head (ALH)
– VCL = Mean distance btw 1st sperm- 2nd sperm position/time
– VSL = Distance btw 1st –last sperm position/time
– VAP = Average path velocity
– ALH = Mean deviation from average path
– LIN = Linearity (VSL/VCL)
– STR = Straightness (VSL/VAP)
Computer Assisted Semen Analysis

18. Computer Assisted Semen Analysis

19. Computer Assisted Semen Analysis

20. • Fructose is a marker of seminal vesicle function.It is an
important energy source for the sperm and its absence
results in immotile sperms.
• DONE WHEN
• The volume <1 ml-seminal vesicle or ejaculatory duct
obstruction.
• Polyzoospermia and low motility.
FRUCTOSE ESTIMATION

21. • Same colour or pink
means fructose
present
• Absecne of colour
means fructose
absent.

25. Immunobead test
• Spermatozoa mixed with
beads coated with IgG class-
specific secondary antibodies
(IgG, IgA, and IgM)
• Antibodies considered
clinically significant when
 50% spermatozoa coated or
• When spermatozoa unable to
penetrate preovulatory human
cervical mucus, or show
impaired fertilizing capacity

27. • Proteolytic enzymes in acrosome digest through zona
pellucida, allowing for sperm–oolemma fusion
• Illustrating this, round-headed sperm, indicative of
acrosomal absence, will not bind to or penetrate through
the zona pellucida
• Staining with different fluorescent lectins that bind to either
the outer membrane or acrosomal contents can assess
the acrosomal integrity
Acrosomal Integrity & function assay

28. Acrosomal Integrity & function assay

29. Gelatin film lysis
test

30. • The precoated gelatin film on the slide gets lysed with
proteolytic enzymes in the acrosome and allows a light to
pass through causing a halo
Principle

31. • Binding of spermatozoa to zona pellucida triggers
acrosome reaction
• Hemizona assay uses human oocytes from which zona
pellucida is isolated & divided in half
– One half is incubated with fertile donor sperm (positive
control) and
– Other half is incubated with patient sperm
– Ratio of fertile to donor binding is measured, with less
than 30% being considered abnormal
Hemizona assay
Arslan M, Morshedi M, Arslan EO et al. Predictive value of the hemizona assay for pregnancy outcome in patients
undergoing controlled ovarian hyperstimulation with intrauterine insemination. Fertil. Steril. 2006; 85: 1697–707

32. Hemizona assay

33. • Uses patient sperm & fertile donor sperm labeled with
different fluorochromes
• Sperm incubated with zona-intact oocytes  number of
bound sperm is counted
• Poor zona binding  correlate with poor fertilization rates
using IVF
• Particularly useful in patients that have failed standard IVF
Sperm zona binding ratio test

34. • HOST (Hyper Osmotic Test):
– Measures acrosin levels
– Include a gelatinolysis technique and a
spectrophotometric assay
• Acrosin  serine protease-like enzyme that exhibits a
lectin-like carbohydrate binding activity to zona pellucida
glycoproteins
• Low acrosin activity associated with low sperm density,
motility, poor normal morphology
Biochemical tests
Xu SP, Zhan BE. Analysis of relationship between semen quality & sperm acrosin activity. Zhonghua Nan Ke Xue 2006;12(5):438–

35. • Citric acid, zinc, alpha glutamyl transferase & acid
phosphate  biochemical substances associated with
prostate
– Have antioxidative properties that neutralize ROS in
seminal plasma
• Zinc  necessary for chromatin stability and
decondensation & role in head– tail detachment in
fertilization
– Measured by colorimetric methods with a reference
value of 13 mmoL per ejaculate
• Reports on effects of zinc in sperm function & semen
Biochemical tests
WHOlaboratory manual for the examination of human semen and sperm–cervical mucus interaction. 4th edition.
Cambridge (UK): Published on behalf of the World Health Organization by Cambridge University Press 1999. p. x, 128

36. • Fructose assay
– Positive correlation between sperm motility and seminal
fructose levels found
• Low or absent fructose seen in ductal obstruction &
congenital absence of vas deferens (CBAVD)
• Karvonen & Malm Method:
– Under influence of heat & low pH, fructose forms a
coloured complex with indole
– Lower reference limit for fructose is 13 mol per
ejaculate
Biochemical tests
Cooper TG et al. (1991). Variations in semen parameters from fathers. Human Reproduction, 6:859-866

37. • L-carnitine  role in sperm maturation
– Low L-carnitine levels found in oligoasthenozoospermic
men
• Alpha glucosidase  tested by fluorimetric methods, has
been used to distinguish nonobstructive from obstructive
azoospermia
– Used as specific marker for epididymal function
– Believed to play a role in sperm maturation in
epididymis
– Cut-off value of 12 mIU/mL distinguishes ductal
Biochemical tests
Comhaire F, et al. Why do we continue to determine alpha-glucosidase in human semen? Andrologia 2002;34(1):8–

38. • ROS in large amounts causes spermatozoal damage by
lipid peroxidation of plasma membrane, germ cell
apoptosis & DNA strand breakage
– Leukocytes or WBCs main source of ROS
–  ROS levels have negative correlation with sperm
concentration, motility, morphology & overall normal
semen parameters
– Smoking, alcohol abuse, and exposure to radiation and
toxic chemical have been associated with increased
seminal ROS
Reactive oxygen species assay

39. • Measurement of ROS is done MC by chemiluminescence
– Measures total seminal ROS (from WBC, abnormal
spermatozoa & seminal fluid
– Leukocytospermia associated with  ROS levels & can
serve as indirect measurement of ROS
– Normal or reference values  0–5.5 104 cpm
• Measurement of oxidative stress (ROS-TAC score)
accurate determination of total effectual ROS
– Score  30can help in prediction of pregnancy
outcomes
Reactive oxygen species assay
Sharma RK, et al. reactive oxygen species–total antioxidant capacity score is a new measure of
oxidative stress to predict male infertility. Hum Reprod 1999;14(11):2801–7.

40. • Protamine deficiency & mutations may affect DNA
packaging or compaction during spermiogenesis
• Factors associated with  sperm DNA damage are:
– Tobacco use,
– Chemotherapy,
– Testicular carcinoma, &
– Other systemic cancers
• DNA damage  correlated positively with poor semen
parameters (8% of subfertile men with normal semen
parameters have high abnormal DNA
DNA fragmentation
Aitken RJ, Irvine DS, Wu FC. Prospective analysis of sperm–oocyte fusion and reactive oxygen
species generation as criteria for the diagnosis of infertility. Am J Obstet Gynecol 1991;164(2):542–51

41. • Indications for DNA fragmentation testing:
• Unexplained or persistent infertility
• Failure to conceive after 5–6 intrauterine insemination
(IUI) cycles despite good count and motility
• Low fertilization rates or poor embryo quality in IVF cycles
• IR failure after IVF
• Recurrent miscarriage
• Prolonged stay in an environment that exposes to
reproductive toxins
• Abnormal semen analysis
• Advancing male age (>45 years).

42. • Direct methods for DNA damage assay include:
– Single cell electrophoresis (COMET)
– Terminal deoxynucleotidyl transferase medicated 2-
deoxyuridine 5-triphosphate (TUNEL)
• Indirect methods:
– Sperm chromatin structure assay (SCSA)
– DNA intercalating dyes (acridine orange)
–Sperm halo test
• Other tests: Sperm DNA denaturation test & sperm
chromatin dispersion test
Sperm DNA fragmentation tests

43. • Single stranded DNA Break (ss-DB)
• Double stranded DNA Break (ds-DB)
• Base deletion or modification
• Inter or Intra-strand cross linkage
Defects in Sperm DNA structure

44. • Environmental Factors:
– Phtalate exposure
– Radiation
– Temperature
• Diseases
– Varicocele
– Genitourinary tract infections
– Fever
• Life-style
– Obesity, Smoking & aging
External factors leading to DNA demage

45. • Terminal deoxynucleotidyl transferese dUTP Nick end
Labeling:
TUNEL

46. • Comet method is based on single cell electrophoresis with
migration of small DNA fragments into a gel and formation
of a “comet”
• This method is also based on a microscopic assessment.
• Number of cells (usually 100) is far too low to satisfy the
need for good counting statistics.
Comet

47. • Sperm DNA fragmentation has little or nothing to do with
parameters that we measure on routine semen analysis
• It has little to do with the shape of the sperm or whether
the sperm are moving. It is a completely independent
variable.
• Men with otherwise normal semen analyses can have a
high degree of DNA damage and men with what was
called very poor sperm quality can have very little DNA
damage.
Sperm chromatin structure assay

48. • Degree of DNA fragmentation correlates very highly with
inability of sperm to initiate a birth regardless of
technology used to fertilize egg such as insemination, IVF
or ICSI.
• Sperm with high DNA fragmentation may fertilize an egg
and embryo development stops before implantation or
may even initiate a pregnancy but there is a significantly
higher likelihood that it will result in miscarriage.
Sperm chromatin structure assay

49. • SPERMS ARE EXPOSED TO STAIN WHICH STAINS ITS
DNA
• THEN ACIDIC TREATMENT MAKES THE SPERM
MEMBRANE LEAKY
• THEN COILED SPERM LEAKS THROUGH MEMBRANE
AS HALO AROUND THE HEAD OF THE SPERM
• THEN READ AS BIG HALO AND MEDIUM HALO AS
NON FRAGMENTED DNA
• WITHOUT HALO AND DEGRADED SPERMS AS
FRAGMENTED
HALO SPERM TEST

52. Electron microscopy
• Ultrastructural details of sperm only can be seen under
electron microscope
• Patients who have low sperm motility ( 5% to 10%) with
high viability (HOST or Eosin-Nigrosin staining) & density
may be appropriate candidates for EM assessment
• Mitochondrial and microtubular defects that are not visible
under the usual Papaniculau smear will be evident
• Subfertile men have more serrated & blurred circular
sulcus, less intact acrosome membrane, a bigger
proportion of spermatic head & more droplets attached to
acrosome membrane
Yu JJ, Xu YM. Ultrastructural defects of acrosome in infertile men. Arch Androl 2004;50(6):405–9

53. EMERGING TECHNOLOGIES

54. • Analyzes transcriptome of cells & tissues
• May provide clues to molecular mechanisms to genetic
infertility (ie, Yq microdeletions)
• Primary application  uncovering the still unknown genes,
pathways, and mechanisms in sperm production
• Creation of mRNA profiles may distinguish spermatogenic
infertility from other causes
Microarray
Ferlin A, et al. Male infertility: role of genetic background. Reprod Biomed Online 2007;14(6):734–45

55. • Main advantage :
– Three-dimensional images (not seen on electron
microscopy)
– Simplicity of sample preparation (air drying)
• Disadvantages:
– Image quality is limited by radius of curvature of probe
tip
– Incorrect tip can result in image artifacts
• Studies in sperm plasma membrane during maturation &
capacitation have identified new areas with
phosphorylated proteins, and large aggregates of lipid did
Atomic force microscopy
Varghese AGet al. Emerging technologies for the molecular study of infertility and potential clinical applications 2007

56. • AT primary care level, a proper diagnosis on male
infertility can be made with:
– Comprehensive and properly performed semen
&
– Thorough history & physical examination
• Advent of new tests should be geared toward better
understanding of intricacies in this haploid cell
• Carefully performed semen analysis remains initial choice
in evaluation of male infertility
• New developments in semen testing promise continued
Summary

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