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SEMEN ANALYSIS: WHO 6TH
EDITION UPDATE
DR. DEEPTHI REPALLE
IVF LAB DIRECTOR
MOHAK IVF
SAIMS
INDORE
Introduction
Introduction
• Sperm numbers, sperm motility
• Sperm morphology
BASIC
EXAMINATIONS
• Leukocytes, immature germ cells, sperm antibodies, indices
of multiple defects
• Biochemical assays, sperm aneuploidy, sperm genetics,
DNA fragmentation
EXTENDED
ANALYSIS
• ROS, Acrosome reaction, sperm chromatin, ion flux
transport
• CASA, Emerging techniques
ADVANCED
EXAMINATION
Introduction
• Sperm numbers, Sperm motility
• Sperm morphology
BASIC
EXAMINATIONS
• Leukocytes, Immature germ cells, Sperm antibodies,
Indices of multiple defects
• Biochemical assays, Sperm aneuploidy, Sperm genetics,
DNA fragmentation
EXTENDED
ANALYSIS
• ROS, Acrosome reaction, Sperm chromatin, Ion flux
transport
• CASA, Emerging techniques
ADVANCED
EXAMINATION

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Introduction
• Basic Examination
Basic examination
Preparations-pre-examination procedures
• Patients information
-Clear written and spoken instructions
-Abstinence- 2-7 days
• Sample collection
-Date and time
-Clinic/home collection
• Sample reception
-Collection difficulty
-Complete collection
• Initial sample handling
Basic examination
• Time vs. examinations
Time -
5min
Volume
liquefaction
Time -60 min
Macroscopic
assessment
Sperm motility
Sperm dilutions
Sperm smears
Time-3 hrs
Sperm
concentration
Time-later
on
Sperm
morphology
Basic examination
Macroscopic examination
Appearance: cream/grey opalescent
Yellowish
Red-brown
Liquefaction: 30-60 min
Viscosity: slightly viscous
Odour: strong odour of urine, putrefaction can be of
clinical importance
Ph: ≥ 7.2

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It was while performing SUZI that a single spermatozoon accidentally penetrated into the oolemma and provided the hint that a direct sperm injection would be more efficient. 1st successful birth by ICSI took place on Jan 14, 1992.

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Basic examination
Microscopic examination
- Phase contrast optics
Under low magnification (100x)
- Sperm aggregation
- Sperm agglutination
- Insufficient liquefaction
Under high magnification (200x/400x)
- Sperm motility
- Dilution required
- Presence of other cells
Non-specific aggregation of
spermatozoa
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Sperm motility
• At least 200 sperms counted in all categories.
• A four-category system for grading motility is
recommended.
• Rapidly progressive ( 25 μm/s) – spermatozoa
moving actively, either linearly or in a large
circle, covering a distance, from the starting
point to the end point, of at least 25 μm (or ½
tail length) in one second.

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Sperm motility
• Slowly progressive (5 to < 25 μm/s) – spermatozoa moving actively,
either linearly or in a large circle, covering a distance, from the
starting point to the end point, of 5 to < 25 μm (or at least one head
length to less than ½ tail length) in one second.
• Non-progressive (< 5 μm/s) – all other patterns of active tail
movements with an absence of progression – i.e. swimming in small
circles, the flagellar force displacing the head less than 5 μm (one
head length), from the starting point to the end point.
• Immotile – no active tail movements.
Sperm motility
Sperm vitality
• Sperm vitality, as estimated by assessing the
membrane integrity of the cells, can be
determined routinely on all ejaculates, but is
not necessary when at least 40% of
spermatozoa are motile.
• In samples with poor motility, the vitality test
is important to discriminate between immotile
dead sperm and immotile live sperm.
Sperm vitality
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• The practical evaluation of human sperm
morphology comprises the following steps:
1. Preparing a smear of ejaculate on a slide
2. Air-drying, fixing and staining the slide
3. Mounting the slide with a coverslip if the slide is
to be kept for a long time
4. Examining the slide with brightfield optics at
×1000 magnification with oil immersion
5. Assessing approximately 200 sperms.
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Reference values
Introduction
• Sperm numbers, sperm motility
• Sperm morphology
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EXAMINTAIONS
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of multiple defects
• Biochemical assays, sperm aneuploidy, sperm genetics,
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ANALYSIS
• ROS, Acrosome reaction, sperm chromatin, ion flux
transport
• CASA, Emerging techniques
ADVANCED
EXAMINATION
Extended analysis
Extended analysis
1. Indices of multiple sperm defects
2. Sperm DNA fragmentation
3. Genetic and genomic tests
4. Tests related to immunology and immunological
methods
5. Assessments of interleukins
6. Assessments of immature germ cells
7. Testing for antibody coating of spermatozoa
8. Biochemical assays for accessory sex glands

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Extended analysis
1. Indices of multiple sperm defects
• The teratozoospermia index (TZI)
• The multiple anomalies index (MAI)
• The sperm deformity index (SDI)
Indices of multiple sperm defects
Sperm DNA fragmentation
• Sperm DNA fragmentation (sDF) is one of the
most common disturbances affecting the genetic
material in the form of single or double strand
breaks.
• sDF may be triggered by different processes,
including the defective packaging of the DNA
during spermatogenesis, and processes of cell
death and oxidative stress which may be
associated with several pathological and
environmental conditions.
Sperm DNA fragmentation
• The techniques of the various SDF tests (TUNEL,
Comet assays, Sperm Chromatin Dispersion test,
and Acridine Orange flow cytometry) have been
described and notes on the clinical interpretation
of these assays have been included. The editors
have also provided suggested thresholds for
clinical decision-making.
• Comet assay should not be used in clinical
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Sperm DNA fragmentation
• The application of sperm DNA tests in clinical
practice remains controversial. This
controversy stems in part from the modest
predictive value of SDF tests in reproduction
and the multitude of available tests with
variable thresholds and inter-lab variability.
Genetic and genomic tests
• Clinically, there is growing awareness that
chromosomal anomalies (numerical,
structural, [including microdeletions and
microduplications]) and gene mutations
underlie a diverse spectrum of male infertility
that underlie many of the anomalies seen in a
semen analysis.
• Sperm aneuploidy test
• FISH
Tests related to immunology and
immunological methods
• Assessments of leukocytes in semen
-Staining cellular peroxidase using ortho-
toluidine
-Panleukocyte (CD45) immunocytochemical
staining
Tests related to immunology and
immunological methods
• The consensus threshold value of 1.0×106
cells/ml for peroxidase-positive cells implies a
higher concentration of total leukocytes, since
not all leukocytes are peroxidase-positive
granulocytes.

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Assessments of interleukins
• Markers of male genital tract inflamation.
• Evaluation of chemokines and cytokines in
semen may be required by andrologists and
urologists to deepen the diagnostic procedure
of infertile males affected by MGT
inflammatory states.
Assessments of immature germ cells
• Germ cells include round spermatids and
spermatocytes, but rarely spermatogonia. They can be
detected in stained semen smears but may be difficult
to distinguish from inflammatory cells when the cells
are degenerating.
• The WHO laboratory manual for the examination of
human semen and sperm cervical mucus interaction,
fourth edition stated that > 6 million immature germ
cells/ml was abnormal. This is no longer provided as a
reference, as no firm evidence base could be found for
this cut-off value.
Testing for antibody coating of the
spermatozoa
• If spermatozoa demonstrate agglutination (i.e.
motile spermatozoa stick to each other head to
head, tail to tail or in a mixed way), the presence
of sperm antibodies is one possible cause to be
investigated.
• Sperm antibodies can be present without sperm
agglutination; equally, agglutination can be
caused by factors others than sperm antibodies.
• The mere presence of sperm antibodies is
insufficient for a diagnosis of sperm
autoimmunity.
Testing for antibody coating of the
spermatozoa
• Tests for antibodies on spermatozoa (“direct
tests”): Two direct tests are described here:
the mixed antiglobulin reaction (MAR) test
and the immunobead (IB) test.
• Tests for ASAB in sperm-free fluids, i.e.
seminal plasma, blood serum and solubilized
cervical mucus (“indirect” tests).

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SPERM FUNCTION TESTS
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SPERM FUNCTION TESTS

Sperm Function Tests are the keystones of evaluating functional condition of sperms. The fertility potential of a sperm will be decided not only with the number & motility but with the functional competence which is of utmost importance.

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Biochemical assays for accessory sex
glands
• Secretory capacity of the prostate.
-zinc, citric acid or acid phosphatase in semen gives
a reliable measure of prostate gland secretion
• Secretory capacity of the seminal vesicles.
-Fructose in semen reflects the secretory function of
the seminal vesicles.
• Secretory capacity of the epididymis.
-L-carnitine, GPC and neutral α-glucosidase are
epididymal markers used clinically.
Introduction
• Sperm numbers, sperm motility
• Sperm morphology
BASIC
EXAMINATIONS
• Leukocytes, immature germ cells, sperm antibodies, indices
of multiple defects
• Biochemical assays, sperm aneuploidy, sperm genetics,
DNA fragmentation
EXTENDED
ANALYSIS
• ROS, Acrosome reaction, sperm chromatin, ion flux
transport
• CASA, Emerging techniques
ADVANCED
EXAMINATION
Advanced examination
Advanced examination
• These tests are generally for research purposes.
1. Seminal oxidative stress and reactive oxygen
species testing
2. Assessment of acrosome reaction
3. Assessment of sperm chromatin
4. Transmembrane ion flux and transport in semen
5. Computer aided sperm analysis (CASA)

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Andrology.pptAndrology.ppt
Andrology.ppt

ANDROLOGY Andrology • Habard Siebke first used the term andrology in 1951, and the field first emerged from dermatology in Germany. • However, urology, gynecology, and endocrinology have a greater impact on modern andrology. • At least 15% of couples throughout the world experience andrological issues, which have become both a prevalent and significant problem. • Male infertility, male contraception, hypogonadism, erectile dysfunction, and male senescence are among the main issues addressed by andrology. • Andrology encompasses a variety of conditions, including testicular cancer, prostate disorders like benign prostatic hyperplasia and carcinoma, delayed puberty, family planning and contraception, cryopreservation of semen and testicular tissue, hormone replacement therapy, forensic paternity issues, and aging in men. Symptomatology of male infertility • TYPE I – erection problems (0,3-7%) • TYPE II – azoospermia (0,9%-16%) • TYPE III – immunological infertility (3,4%-25%) • TYPE IV – abnormal seminal quality (23%-48%) • TYPE V – idiopathic sperm dysfunction (0-25%) Diagnosis • General examination • Semen analysis • Other diagnostic tests: • USG • Hormonal diagnostic • Diagnostic tests for Assisted Reproductive Technology TYPE I – erection problems (0,3-7%) • Normal ejaculation • Hypospermia (semen volume < 2,0 ml) – chronic prostatitis • Impotence • Retrograde ejaculation • Neurogenic– DM, SM • Anatomical • Jatrogenic – drugs, operations • disejaculation • Functional – anorgazmia • Neurogenic – spinal injury • Jatrogenic – drugs, chemiotherapy, radiotherapy, operations TYPE II – azoospermia (0,9%-16%) • Pre-testicular causes • Hypothalamic or pituitary disorder – LH, FSH deficiency, Kallman syndrome, trauma, tumors, inflammation, meningitis • Testicular causes • Primary testicular failure • Congenital – 47XXY, del Y, AZF • Acquired- mumps, testicular torsion, castration • Jatrogenic – radiotherapy, chemotherapy • Post-testicular causes • Congenital • Acquired – inflammations (gonorrhea) • Jatrogenic – vasectomy, hernia operation Diagnostic tests for Assisted Reproductive Technology- ICSI • FSH • If < 12IU – sperm biopsy is effective in 80-90% • Blocked ejaculatory duct (Micro-Epididymal Sperm Aspiration –MESE) • Other (Testicular Sperm Extirpation- TESE, Testicular Sperm Aspiration- TESA) TYPE III – immunological infertility (3,4%-25%) Antisperm antibodies – the immune system may produce antibodies that attack and weaken or disable sperm • Auto-immunological diseases • Consequences of testicular trauma Congenital • Undescended testicles Sexually transmitted disease (gonorrhoea) or testicular infection (mumps) • Vascular Testicular torsion • Varicocoeles Diseases: Thyroid failure; Addison disease. • auto-immunological diseases; • Environmental factors Drugs (sulfasalazine, T, chemotherapy) • Temperature Other factors (X-rays, lead, cigarette s

andrologyreproductive physiology
Seminal oxidative stress and reactive
oxygen species testing
• Reactive oxygen species (ROS) produced by
leukocytes underlie their deleterious effects
when present at high level in semen.
1. Luminol
2. Oxidation-reduction potential
3. Total antioxidant capacity
Assessment of the acrosome reaction
Assessment of the acrosome reaction
Assessment of the acrosome reaction
• The integrity of the acrosome structure and the ability to undergo
acrosomal exocytosis are necessary for normal fertility.
• The acrosome reaction is a process that in vivo occurs in the
proximity of the oocyte, and which must take place before the
spermatozoon can penetrate the oocyte vestments and fuse with
the oocyte.
• Calcium influx is believed to be an initiating event in the normal
acrosome reaction.
• In cases of teratozoospermia and oligozoospermia, some patients
may have otherwise normal results of ejaculate examination, but
spermatozoa may display alterations in the acrosomal structure or
in the ability to respond to stimuli of acrosome reaction

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This document discusses sperm sorting techniques for men with high sperm DNA fragmentation index. It begins by defining sperm DNA damage and fragmentation, then discusses causes and indications for testing. It describes different tests to measure sperm DNA fragmentation and diagnostic cut-off points. The objectives and limitations of sperm sorting are outlined. Various sperm sorting techniques are explained, including swim-up, density gradient, magnetic activated cell sorting (MACS), motile sperm organelle morphology examination (MSOME), and surface charge-based sorting using hyaluronan binding. Advanced techniques like MSOME and MACS aim to select sperm with intact DNA and normal morphology to improve fertility outcomes.

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Search for Dark Matter Ionization on the Night Side of Jupiter with Cassini
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We present a new search for dark matter (DM) using planetary atmospheres. We point out that annihilating DM in planets can produce ionizing radiation, which can lead to excess production of ionospheric Hþ 3 . We apply this search strategy to the night side of Jupiter near the equator. The night side has zero solar irradiation, and low latitudes are sufficiently far from ionizing auroras, leading to a lowbackground search. We use Cassini data on ionospheric Hþ 3 emission collected three hours either side of Jovian midnight, during its flyby in 2000, and set novel constraints on the DM-nucleon scattering cross section down to about 10−38 cm2. We also highlight that DM atmospheric ionization may be detected in Jovian exoplanets using future high-precision measurements of planetary spectra.

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El Nuevo Cohete Ariane de la Agencia Espacial Europea-6_Media-Kit_english.pdf
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Europe must have autonomous access to space to realise its ambitions on the world stage and promote knowledge and prosperity. Space is a natural extension of our home planet and forms an integral part of the infrastructure that is vital to daily life on Earth. Europe must assert its rightful place in space to ensure its citizens thrive. As the world’s second-largest economy, Europe must ensure it has secure and autonomous access to space, so it does not depend on the capabilities and priorities of other nations. Europe’s longstanding expertise in launching spacecraft and satellites has been a driving force behind its 60 years of successful space cooperation. In a world where everyday life – from connectivity to navigation, climate and weather – relies on space, the ability to launch independently is more important than ever before. With the launch of Ariane 6, Europe is not just sending a rocket into the sky, we are asserting our place among the world’s spacefaring nations. ESA’s Ariane 6 rocket succeeds Ariane 5, the most dependable and competitive launcher for decades. The first Ariane rocket was launched in 1979 from Europe’s Spaceport in French Guiana and Ariane 6 will continue the adventure. Putting Europe at the forefront of space transportation for nearly 45 years, Ariane is a triumph of engineering and the prize of great European industrial and political cooperation. Ariane 1 gave way to more powerful versions 2, 3 and 4. Ariane 5 served as one of the world’s premier heavy-lift rockets, putting single or multiple payloads into orbit – the cargo and instruments being launched – and sent a series of iconic scientific missions to deep space. The decision to start developing Ariane 6 was taken in 2014 to respond to the continued need to have independent access to space, while offering efficient commercial launch services in a fast-changing market. ESA, with its Member States and industrial partners led by ArianeGroup, is developing new technologies for new markets with Ariane 6. The versatility of Ariane 6 adds a whole new dimension to its very successful predecessors

nuevo cohete ariane 6 - esa
Assessment of sperm chromatin
• The stability of the sperm chromatin structure
is of fundamental importance for embryo
development and quality, probably due to
protection of and rapid availability of the
paternal genome.
1. Aniline blue assessment
2. Chromomycin A3 assessment
Transmembrane ion flux and transport
in sperm
• Diagnostic tools to better understand male
factor infertility and disorders of the male
reproductive organs.
1. Electrophysiology and kinetic Ca2+
fluorimetry to assess the function of CatSper
2. Electrophysiological and fluorimetric
methods to study the function of K+ channels
3. Methods to detect (mal)-function of ion
transporters and exchangers
Computer-aided sperm analysis (CASA)
• Using CASA to assess sperm motility
CASA terminology
• VCL, velocity along the curvilinear path (μm/s)
• VSL, velocity along the straight-line path (μm/s)
• VAP, velocity along the average path (μm/s)
• ALH, the amplitude of the lateral displacement of the head (μm)
• MAD, mean angular displacement (degrees)
• Automated systems may be useful for obtaining additional research
data (including on morphological subpopulations of spermatozoa,
plasma membrane integrity, sperm energy index) and for QC
systems, but more research is needed to show their benefits for
clinical purposes.
Significance
• The reference ranges described in the 5th
edition should be abandoned as they are of
limited value in differentiating fertile and
infertile man.
• Who reference ranges did not adequately
reflect fertility dynamics of the male partner.
• 5th percentile is commonly used as a statistical
approach to determine cut-off norms in
medical tests.

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The rapid assembly of the first supermassive black holes is an enduring mystery. Until now, it was not known whether quasar ‘feeding’ structures (the ‘hot torus’) could assemble as fast as the smaller-scale quasar structures. We present JWST/MRS (rest-frame infrared) spectroscopic observations of the quasar J1120+0641 at z = 7.0848 (well within the epoch of reionization). The hot torus dust was clearly detected at λrest ≃ 1.3 μm, with a black-body temperature of  K, slightly elevated compared to similarly luminous quasars at lower redshifts. Importantly, the supermassive black hole mass of J1120+0641 based on the Hα line (accessible only with JWST), MBH = 1.52 ± 0.17 × 109 M⊙, is in good agreement with previous ground-based rest-frame ultraviolet Mg II measurements. Comparing the ratios of the Hα, Paα and Paβ emission lines to predictions from a simple one-phase Cloudy model, we find that they are consistent with originating from a common broad-line region with physical parameters that are consistent with lower-redshift quasars. Together, this implies that J1120+0641’s accretion structures must have assembled very quickly, as they appear fully ‘mature’ less than 760 Myr after the Big Bang.

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Keys of Identification for Indian Wood: A Seminar Report
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Identifying Indian wood involves recognizing key characteristics such as grain patterns, color, texture, hardness, and specific anatomical features. These identification keys include observing the wood's pores, growth rings, and resin canals, as well as its scent and weight. Understanding these features is essential for accurate wood identification, which is crucial for various applications in carpentry, furniture making, and conservation. Additionally, the application of Convolutional Neural Networks (CNN) in wood identification has revolutionized this field. CNNs can analyze images of wood samples to identify species with high accuracy by learning and recognizing intricate patterns and features. This technological advancement not only enhances the precision of wood identification but also accelerates the process, making it more efficient for industry professionals and researchers alike.

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Properties of virus(Ultrastructure and types of virus)
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It is obligate type of parasite which affect living organism.

microbes
WHO 6TH EDITION UPDATE FOR SEMEN ANALYSIS
WHO 6TH EDITION UPDATE FOR SEMEN ANALYSIS
5th edition vs. 6th edition
Decision limits
• Reference ranges were replaced by the
decision limits in the 6th edition.
1. Normal
2. Borderline
3. Pathological

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A slightly oblate dark matter halo revealed by a retrograde precessing Galact...
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The shape of the dark matter (DM) halo is key to understanding the hierarchical formation of the Galaxy. Despite extensive eforts in recent decades, however, its shape remains a matter of debate, with suggestions ranging from strongly oblate to prolate. Here, we present a new constraint on its present shape by directly measuring the evolution of the Galactic disk warp with time, as traced by accurate distance estimates and precise age determinations for about 2,600 classical Cepheids. We show that the Galactic warp is mildly precessing in a retrograde direction at a rate of ω = −2.1 ± 0.5 (statistical) ± 0.6 (systematic) km s−1 kpc−1 for the outer disk over the Galactocentric radius [7.5, 25] kpc, decreasing with radius. This constrains the shape of the DM halo to be slightly oblate with a fattening (minor axis to major axis ratio) in the range 0.84 ≤ qΦ ≤ 0.96. Given the young nature of the disk warp traced by Cepheids (less than 200 Myr), our approach directly measures the shape of the present-day DM halo. This measurement, combined with other measurements from older tracers, could provide vital constraints on the evolution of the DM halo and the assembly history of the Galaxy.

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Protein Digestion123334444556678890.pptx

PROTEIN DIgestion

pepainpolypeptideaprotease
Decision limits
Characteristics units Normal Borderline Pathological
Volume ML 2-6 1.5-1.9 <1.5
Sperm
concentration
Million/ML ≥20 10-20 <10
Total sperm
count
Million/ejaculate ≥80 20-79 <20
Motility % of motile ≥60 40-59 <40
% of progressive ≥50 35-49 <35
% rapid
progressive
≥25
Morphology % typical forms ≥14 4-13 <4
Vitality % of live ≥60 40-59 <40
Conclusions
• The 6th edition is a step forward in our understanding
of the complex subject of male infertility through
evaluation of the human ejaculate.
• Several objections to the 5th edition have been
addressed in the 6th edition, though areas of
controversy remain.
• For the clinician, the most notable change is the
introduction of “decision limits” rather than reference
ranges, and the endorsement of SDF as an extended
seminal test that can be ordered in certain clinical
situations.
Conclusions
• For the laboratory personnel, the manual has
been streamlined to facilitate a step-by-step
examination with several old tests being
abandoned, while several new tests have been
introduced.
• The need for the extended analysis has been
explained in detailed.
• Advanced and emerging technologies are
included for research purposes.
HISTORY FORM

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WHO 6TH EDITION UPDATE FOR SEMEN ANALYSIS

  • 1. SEMEN ANALYSIS: WHO 6TH EDITION UPDATE DR. DEEPTHI REPALLE IVF LAB DIRECTOR MOHAK IVF SAIMS INDORE
  • 3. Introduction • Sperm numbers, sperm motility • Sperm morphology BASIC EXAMINATIONS • Leukocytes, immature germ cells, sperm antibodies, indices of multiple defects • Biochemical assays, sperm aneuploidy, sperm genetics, DNA fragmentation EXTENDED ANALYSIS • ROS, Acrosome reaction, sperm chromatin, ion flux transport • CASA, Emerging techniques ADVANCED EXAMINATION
  • 4. Introduction • Sperm numbers, Sperm motility • Sperm morphology BASIC EXAMINATIONS • Leukocytes, Immature germ cells, Sperm antibodies, Indices of multiple defects • Biochemical assays, Sperm aneuploidy, Sperm genetics, DNA fragmentation EXTENDED ANALYSIS • ROS, Acrosome reaction, Sperm chromatin, Ion flux transport • CASA, Emerging techniques ADVANCED EXAMINATION
  • 6. Basic examination Preparations-pre-examination procedures • Patients information -Clear written and spoken instructions -Abstinence- 2-7 days • Sample collection -Date and time -Clinic/home collection • Sample reception -Collection difficulty -Complete collection • Initial sample handling
  • 7. Basic examination • Time vs. examinations Time - 5min Volume liquefaction Time -60 min Macroscopic assessment Sperm motility Sperm dilutions Sperm smears Time-3 hrs Sperm concentration Time-later on Sperm morphology
  • 8. Basic examination Macroscopic examination Appearance: cream/grey opalescent Yellowish Red-brown Liquefaction: 30-60 min Viscosity: slightly viscous Odour: strong odour of urine, putrefaction can be of clinical importance Ph: ≥ 7.2
  • 9. Basic examination Microscopic examination - Phase contrast optics Under low magnification (100x) - Sperm aggregation - Sperm agglutination - Insufficient liquefaction Under high magnification (200x/400x) - Sperm motility - Dilution required - Presence of other cells
  • 12. Sperm motility • At least 200 sperms counted in all categories. • A four-category system for grading motility is recommended. • Rapidly progressive ( 25 μm/s) – spermatozoa moving actively, either linearly or in a large circle, covering a distance, from the starting point to the end point, of at least 25 μm (or ½ tail length) in one second.
  • 13. Sperm motility • Slowly progressive (5 to < 25 μm/s) – spermatozoa moving actively, either linearly or in a large circle, covering a distance, from the starting point to the end point, of 5 to < 25 μm (or at least one head length to less than ½ tail length) in one second. • Non-progressive (< 5 μm/s) – all other patterns of active tail movements with an absence of progression – i.e. swimming in small circles, the flagellar force displacing the head less than 5 μm (one head length), from the starting point to the end point. • Immotile – no active tail movements.
  • 15. Sperm vitality • Sperm vitality, as estimated by assessing the membrane integrity of the cells, can be determined routinely on all ejaculates, but is not necessary when at least 40% of spermatozoa are motile. • In samples with poor motility, the vitality test is important to discriminate between immotile dead sperm and immotile live sperm.
  • 18. Sperm count • The haemocytometer with improved neubauer ruling
  • 20. Sperm count • Sperm concentration per ml • Total number of spermatozoa per ejaculate • Formula, C=(N/n) x (1/v) x dilution factor.
  • 21. Sperm Morphology • The practical evaluation of human sperm morphology comprises the following steps: 1. Preparing a smear of ejaculate on a slide 2. Air-drying, fixing and staining the slide 3. Mounting the slide with a coverslip if the slide is to be kept for a long time 4. Examining the slide with brightfield optics at ×1000 magnification with oil immersion 5. Assessing approximately 200 sperms.
  • 30. Introduction • Sperm numbers, sperm motility • Sperm morphology BASIC EXAMINTAIONS • Leukocytes, immature germ cells, sperm antibodies, indices of multiple defects • Biochemical assays, sperm aneuploidy, sperm genetics, DNA fragmentation EXTENDED ANALYSIS • ROS, Acrosome reaction, sperm chromatin, ion flux transport • CASA, Emerging techniques ADVANCED EXAMINATION
  • 32. Extended analysis 1. Indices of multiple sperm defects 2. Sperm DNA fragmentation 3. Genetic and genomic tests 4. Tests related to immunology and immunological methods 5. Assessments of interleukins 6. Assessments of immature germ cells 7. Testing for antibody coating of spermatozoa 8. Biochemical assays for accessory sex glands
  • 33. Extended analysis 1. Indices of multiple sperm defects • The teratozoospermia index (TZI) • The multiple anomalies index (MAI) • The sperm deformity index (SDI)
  • 34. Indices of multiple sperm defects
  • 35. Sperm DNA fragmentation • Sperm DNA fragmentation (sDF) is one of the most common disturbances affecting the genetic material in the form of single or double strand breaks. • sDF may be triggered by different processes, including the defective packaging of the DNA during spermatogenesis, and processes of cell death and oxidative stress which may be associated with several pathological and environmental conditions.
  • 36. Sperm DNA fragmentation • The techniques of the various SDF tests (TUNEL, Comet assays, Sperm Chromatin Dispersion test, and Acridine Orange flow cytometry) have been described and notes on the clinical interpretation of these assays have been included. The editors have also provided suggested thresholds for clinical decision-making. • Comet assay should not be used in clinical practice because of an important degree of inter- laboratory variation.
  • 37. Sperm DNA fragmentation • The application of sperm DNA tests in clinical practice remains controversial. This controversy stems in part from the modest predictive value of SDF tests in reproduction and the multitude of available tests with variable thresholds and inter-lab variability.
  • 38. Genetic and genomic tests • Clinically, there is growing awareness that chromosomal anomalies (numerical, structural, [including microdeletions and microduplications]) and gene mutations underlie a diverse spectrum of male infertility that underlie many of the anomalies seen in a semen analysis. • Sperm aneuploidy test • FISH
  • 39. Tests related to immunology and immunological methods • Assessments of leukocytes in semen -Staining cellular peroxidase using ortho- toluidine -Panleukocyte (CD45) immunocytochemical staining
  • 40. Tests related to immunology and immunological methods • The consensus threshold value of 1.0×106 cells/ml for peroxidase-positive cells implies a higher concentration of total leukocytes, since not all leukocytes are peroxidase-positive granulocytes.
  • 41. Assessments of interleukins • Markers of male genital tract inflamation. • Evaluation of chemokines and cytokines in semen may be required by andrologists and urologists to deepen the diagnostic procedure of infertile males affected by MGT inflammatory states.
  • 42. Assessments of immature germ cells • Germ cells include round spermatids and spermatocytes, but rarely spermatogonia. They can be detected in stained semen smears but may be difficult to distinguish from inflammatory cells when the cells are degenerating. • The WHO laboratory manual for the examination of human semen and sperm cervical mucus interaction, fourth edition stated that > 6 million immature germ cells/ml was abnormal. This is no longer provided as a reference, as no firm evidence base could be found for this cut-off value.
  • 43. Testing for antibody coating of the spermatozoa • If spermatozoa demonstrate agglutination (i.e. motile spermatozoa stick to each other head to head, tail to tail or in a mixed way), the presence of sperm antibodies is one possible cause to be investigated. • Sperm antibodies can be present without sperm agglutination; equally, agglutination can be caused by factors others than sperm antibodies. • The mere presence of sperm antibodies is insufficient for a diagnosis of sperm autoimmunity.
  • 44. Testing for antibody coating of the spermatozoa • Tests for antibodies on spermatozoa (“direct tests”): Two direct tests are described here: the mixed antiglobulin reaction (MAR) test and the immunobead (IB) test. • Tests for ASAB in sperm-free fluids, i.e. seminal plasma, blood serum and solubilized cervical mucus (“indirect” tests).
  • 45. Biochemical assays for accessory sex glands • Secretory capacity of the prostate. -zinc, citric acid or acid phosphatase in semen gives a reliable measure of prostate gland secretion • Secretory capacity of the seminal vesicles. -Fructose in semen reflects the secretory function of the seminal vesicles. • Secretory capacity of the epididymis. -L-carnitine, GPC and neutral α-glucosidase are epididymal markers used clinically.
  • 46. Introduction • Sperm numbers, sperm motility • Sperm morphology BASIC EXAMINATIONS • Leukocytes, immature germ cells, sperm antibodies, indices of multiple defects • Biochemical assays, sperm aneuploidy, sperm genetics, DNA fragmentation EXTENDED ANALYSIS • ROS, Acrosome reaction, sperm chromatin, ion flux transport • CASA, Emerging techniques ADVANCED EXAMINATION
  • 48. Advanced examination • These tests are generally for research purposes. 1. Seminal oxidative stress and reactive oxygen species testing 2. Assessment of acrosome reaction 3. Assessment of sperm chromatin 4. Transmembrane ion flux and transport in semen 5. Computer aided sperm analysis (CASA)
  • 49. Seminal oxidative stress and reactive oxygen species testing • Reactive oxygen species (ROS) produced by leukocytes underlie their deleterious effects when present at high level in semen. 1. Luminol 2. Oxidation-reduction potential 3. Total antioxidant capacity
  • 50. Assessment of the acrosome reaction
  • 51. Assessment of the acrosome reaction
  • 52. Assessment of the acrosome reaction • The integrity of the acrosome structure and the ability to undergo acrosomal exocytosis are necessary for normal fertility. • The acrosome reaction is a process that in vivo occurs in the proximity of the oocyte, and which must take place before the spermatozoon can penetrate the oocyte vestments and fuse with the oocyte. • Calcium influx is believed to be an initiating event in the normal acrosome reaction. • In cases of teratozoospermia and oligozoospermia, some patients may have otherwise normal results of ejaculate examination, but spermatozoa may display alterations in the acrosomal structure or in the ability to respond to stimuli of acrosome reaction
  • 53. Assessment of sperm chromatin • The stability of the sperm chromatin structure is of fundamental importance for embryo development and quality, probably due to protection of and rapid availability of the paternal genome. 1. Aniline blue assessment 2. Chromomycin A3 assessment
  • 54. Transmembrane ion flux and transport in sperm • Diagnostic tools to better understand male factor infertility and disorders of the male reproductive organs. 1. Electrophysiology and kinetic Ca2+ fluorimetry to assess the function of CatSper 2. Electrophysiological and fluorimetric methods to study the function of K+ channels 3. Methods to detect (mal)-function of ion transporters and exchangers
  • 55. Computer-aided sperm analysis (CASA) • Using CASA to assess sperm motility CASA terminology • VCL, velocity along the curvilinear path (μm/s) • VSL, velocity along the straight-line path (μm/s) • VAP, velocity along the average path (μm/s) • ALH, the amplitude of the lateral displacement of the head (μm) • MAD, mean angular displacement (degrees) • Automated systems may be useful for obtaining additional research data (including on morphological subpopulations of spermatozoa, plasma membrane integrity, sperm energy index) and for QC systems, but more research is needed to show their benefits for clinical purposes.
  • 56. Significance • The reference ranges described in the 5th edition should be abandoned as they are of limited value in differentiating fertile and infertile man. • Who reference ranges did not adequately reflect fertility dynamics of the male partner. • 5th percentile is commonly used as a statistical approach to determine cut-off norms in medical tests.
  • 59. 5th edition vs. 6th edition
  • 60. Decision limits • Reference ranges were replaced by the decision limits in the 6th edition. 1. Normal 2. Borderline 3. Pathological
  • 61. Decision limits Characteristics units Normal Borderline Pathological Volume ML 2-6 1.5-1.9 <1.5 Sperm concentration Million/ML ≥20 10-20 <10 Total sperm count Million/ejaculate ≥80 20-79 <20 Motility % of motile ≥60 40-59 <40 % of progressive ≥50 35-49 <35 % rapid progressive ≥25 Morphology % typical forms ≥14 4-13 <4 Vitality % of live ≥60 40-59 <40
  • 62. Conclusions • The 6th edition is a step forward in our understanding of the complex subject of male infertility through evaluation of the human ejaculate. • Several objections to the 5th edition have been addressed in the 6th edition, though areas of controversy remain. • For the clinician, the most notable change is the introduction of “decision limits” rather than reference ranges, and the endorsement of SDF as an extended seminal test that can be ordered in certain clinical situations.
  • 63. Conclusions • For the laboratory personnel, the manual has been streamlined to facilitate a step-by-step examination with several old tests being abandoned, while several new tests have been introduced. • The need for the extended analysis has been explained in detailed. • Advanced and emerging technologies are included for research purposes.