SPERMATOGENESIS, SEMEN ANALYSIS AND ANTISPERM ANTIBODIES

SPERMATOGENESIS
SEMEN ANALYSIS
ANTISPERM ANTIBODIES
Dept of Urology
Govt Royapettah Hospital and Kilpauk Medical College
Chennai
1
Dept of Urology, GRH and KMC, Chennai.

Moderators:
Professors:
• Prof. Dr. G. Sivasankar, M.S., M.Ch.,
• Prof. Dr. A. Senthilvel, M.S., M.Ch.,
Asst Professors:
• Dr. J. Sivabalan, M.S., M.Ch.,
• Dr. R. Bhargavi, M.S., M.Ch.,
• Dr. S. Raju, M.S., M.Ch.,
• Dr. K. Muthurathinam, M.S., M.Ch.,
• Dr. D. Tamilselvan, M.S., M.Ch.,
• Dr. K. Senthilkumar, M.S., M.Ch.
Dept of Urology, GRH and KMC, Chennai. 2

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Maturation arrest
Numerous spermatogonia, few
spermatocytes and no mature
spermatozoa.
10

SOCS
Causes of Sertoli cell-only
syndrome include:
Gonadotropin deficiency,
Cryptorchidism,
Viral orchitis,
Irradiation, alkylating agents,
Hormonal therapy for prostate
cancer etc
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Klinefelter syndrome
This testicular biopsy is from an
adult male with history of
normal semen volume and
severe oligospermia. He had
small firm testes and body
habitus suggestive of
Klinefelter’s syndrome. The
biopsy shows small hyalinized
seminiferous tubules and
pseudo-adenomatous clusters of
leydig cells
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SPERMIOGENESIS
Spermiogenesis refer to the differentiation of the
spermatid into the functional spermatozoan.
Spermatid: Non-motile, round, non-specialized
Spermatozoan: Motile, elongate.
Events of Spermiogenesis
 Nuclear Shaping & Condensation.
 Formation of flagellum.
 Formation of Acrosome.
 Shedding of the Residual Body.
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Stored , Matured & Attain motility in epididymis
Capacitation in female genital tract
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Semen analysis
Plays a key role in evaluation of men
presenting with infertility.
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Semen analysis
Tubal patency
Ovulation
Mandatory before any intervention
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What is the purpose of the test?
Investigation of infertility ( Primary or Secondary)
Identify treatment options
Surgical treatment.
Medical treatment.
Assisted conception treatment.
Determine the suitability of semen for ICSI/IVF.
 Pre and Post vasectomy – Confirmation.
 Following vasectomy reversal.
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Human sperm cell is about 70 µm long.
The head size: 4-5µm
Nucleus is in the contains the 23 chromosomes.
Mid-piece: 4-5µm
The energy for motility is generated.
Tail: 55µm
Motility beat - Midpiece - Propagated along the tail.
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FRACTION OF SEMEN CONTRIBUTED BY VARIOUS GLANDS
1. Urethral glands (2-5%) are very small mucus secreting glands.
2. Prostate: Approximately 20-30% of the semen volume is acidic fluid produced by the
prostate gland, the secretion contains citrate, zinc, acid phosphatase and proteolytic
enzymes liquefaction of the semen.
3. Seminal vesicles (produce about 46-80 % of the fluid volume of semen) viscous,
yellowish secretion is rich in fructose, vitamin C, prostaglandin, protein kinase, and
other substances, which nourish and activate the sperm passing through the tract.
4. Testis & Epididymis: (5%) Spermatozoa are produced in the testis under the influence
of testosterone, and then the epididymis (is the first part of the duct system) provides a
temporary storage site for the immature sperm that enter it from testis.
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Standard guidelines for the collection of semen
 There should be 2 to 7 days of sexual abstinence before collection.
 Two separate samples at least 7 days apart should be analyzed.
 The duration of abstinence should be constant
 Masturbation in a clinical setting is the recommended procedure.
 Collection - Private room in the same centre where the semen will be analyzed.
 Pre warmed (21oC), sterile, non-toxic, wide-mouth container.
2 to 7 days 7 days
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PRECAUTIONS
 Abstinence for 2-7 days
 Pass urine
 Wash hands with soap and dry
 Glans and the penis should be cleaned with a wet paper towel (avoid soap).
 Lubricants should be avoided - interfere with motility - a
 A Collect the entire sample -70% of sperms is in the first part of the ejaculate.
Other methods of collection
Coitus interruptus
Condom collection
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Assistance - unable to achieve adequate erection and ejaculation.
Phosphodiesterase type 5 inhibitors - 30 to 60 min before collection.
Cavernosal and subcutaneous injections of prostaglandins
Vacuum erection devices can also be used to obtain erection by creating a vacuum around the
penis, generating a pressure differential that fills the corpora with blood.
Vibratory stimulation may be used for patients who have suffered spinal cord injury, if the spinal
cord lesion is T8 and above.
Rectal probe electro-stimulation induces ejaculation by stimulation of the efferent fibers of the
hypogastric plexus.
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LABEL OF SAMPLE
Patient name
Age
Clinic or Doctor name
Date and time
Laboratory analysis form:
 The period of abstinence (in days).
 Time of collection.
 Complete or incomplete.
 The time interval from collection to analysis.
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STORAGE TIMING OF ANALYSIS
In order to allow liquefaction and mixing,
Semen is placed in a 37° C gently shaking incubator for 30 minutes.
The semen sample should be examined,
Ideally within 30 mins
Absolutely within 1 hour of collection.
Motility decreases significantly after 2 hours
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WHO 2010
Fifth edition (30 years from 1st edition, 1980)
Reference ranges are derived from 4500 SA
Raw data on from recent fathers in 14 countries on four
continents. Chinese data on 429 SA and 4 Singaporean SA was
included. No Indians and other Asian nationalities.
The 5th percentile is given as the lower reference range
No high reference range is given
Ref : Cooper TG et al. Human Reproduction Update, Vol.16, No.3 pp. 231–245, 2010
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Parameter Lower Reference Limit
Semen volume 1.5 ml
Sperm concentration 15 x 106/ml
Total sperm number 39 x106/ejaculate
Progressive motility 32 % A
Total motility 40 % A+B
Vitality (live sperms) 58 %
Sperm morphology 4 %
pH >/=7.2
Leucocyte <1 x106/ml
MAR/Immunobead test <50 %
WHO 2010
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Terminologies in SA (WHO)
Normospermia - Normal semen volume
Aspermia - No semen volume
Hypospermia - Semen volume < 1.5 ml
Hyperspermia - Semen volume > 6.0 ml
Azoospermia - No spermatozoa in semen
Oligospermia - Sperm concentration <15 M/ml
Polyzoospermia - High sperm concentration, >200M/ml
Asthenozoospermia - <40% grade (A&B) or < 32 PR%
Teratozoospermia - <4% spermatozoa
Leukospermia - Leukocytes present in semen, >1M/ml
Hematospermia - Red blood cell present in semen
Necrozoospermia - “dead” sperm
OAT =Oligo-astheno-teratozoospermia 33

WET SMEAR PREPARATION
Normally 10 ul semen to 190 ul water = 20x dilution.
In cases of very low sperm count = 4x dilution
In cases of azoospermia = no dilution
Add 10 ul of mixture to the chamber
Cover slip
Wait 2-3 min to settle
Sum of 5 squares = sperm density x 106/ml
Count only whole sperms, not pinheads
Use the “L” rule or “SS” pattern
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The semen analysis characteristics can be classified into two groups.
Macroscopic
Microscopic
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Volume Normal: 1.5 ml per ejaculation
Low volume (<1ml) reflect a problem with the seminal vesicles
and prostate – a block, retrograde ejaculation, infection or lack
of androgen.
pH Normal: =/>7.2 (alkaline)
Acidic pH (<7.0) in a low volume indicates –congenital
bilateral absence of vas deferens (in which seminal vesicles are
also poorly developed) and ejaculatory duct obstruction.
Macroscopic Examination
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Macroscopic Examination…cont
WHO criteria 2010 Description
Appearance Normal: Whitish to grey opalescent
Yellow (urine, jaundice); Pink/Reddish/Brown (RBCs)
Liquefaction Normal: 15–30 minutes after collection
Lumpy >60 min – sperms may be trapped in unliquefied jelly;
maybe sign of prostatic infection, lack of prostatic protease
Viscosity Normal Smooth and watery
Abnormal thick with long threads.
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Microscopic assessment of semen
Sperm agglutination
Count and concentration
Motility
Morphology
Viability
Nonsperm cells
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SPERM AGGLUTINATION
The microscopic examination of wet smear
Sperm form clumps within semen
Sperm-to-nonsperm elements (nonspecific agglutination) - accessory gland infection.
Sperm-to-sperm agglutination (site-specific agglutination) - antisperm antibodies.
When agglutination is observed - semen cultures and antibody assessment.
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COUNT AND CONCENTRATION.
Sperm concentration (number of sperm per milliliter)
Sperm count (number of sperm per ejaculate)
Azoospermia (absence of sperm)
Abnormal spermatogenesis, ejaculatory dysfunction, or obstruction.
Centrifuged and the pellet examined for the presence of any sperm.
Oligospermia (abnormally lower sperm concentration)
Polyzoospermia (abnormally elevated sperm concentration)- rare.
May be caused by a long period of abstinence - associated with sperm of poor quality.
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MOTILITY
Most important predictor of the functional aspect of spermatozoa.
Sperm motility is a reflection of the normal development of the axoneme.
Sperm motility is a reflection of the normal maturation within the epididymis.
The sperm motility is graded according to the WHO as follows:
A—Rapid forward progress motility;
B—Slow or sluggish progressive motility;
C—Nonprogressive motility;
D—Immotility.
The cutoff value for normal
32% grade A motility
40% grade A+B
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Limitation of sperm motility assessment
The method most commonly employed is the simple estimation of the motility of sperm
on several fields.
Assessment of this parameter is subjective - potential for technical mistakes.
In-vitro motility of sperm may not reflect the true motility within the female
reproductive tract.
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Causes of asthenospermia
Inherent defects of sperm,
Artifactual - Spermicides, Lubricants, Or Rubber Condoms.
Prolonged Abstinence Periods,
Genital Tract Infection,
Partial Ductal Obstruction,
Varicocele.
ASA - peculiar shaking pattern – preventing penetration through cervical mucus.
Occasional clumps of agglutinated sperm are of no consequence.
> 10% to 15% of clumping of spermatozoa is indicative of antisperm antibodies
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MORPHOLOGY
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Viability
When the motility is reported as less than 5% to 10%
To differentiate immotile from dead sperm
 Staining method (commonly used)
 Hypo-osmotic swelling test (HOST) (alternative)
Staining method (commonly used)
Eosin Y followed by counter staining with Nigrosin.
Principle is that viable sperm have intact cell membranes.
Do not take up the dye and will remain unstained.
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Hypo-osmotic swelling test (HOST) (alternative)
Exposure of the sperm to hypoosmotic fluid.
Principle is that viable sperm have intact cell membranes.
Cause swelling of the cytoplasmic space and curling of the sperm tail.
Nonviable sperm - will not exhibit this effect.
Reproducible and relatively inexpensive test
Helps in selection of viable sperm - IVF or ICSI.
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NONSPERM CELLS
Leukocytes: normally (1-4/HPF)
Leukocytospermia as levels above 1 × 106 WBC/mL - infection
Endtz test – reaction with peroxide – ortho-toluidine dye
Epithelial cells: normally (1-2/HPF)
Spermatocytes: (Immature germ cells) 1-2/HPF
Erythrocytes: (1-2/HPF). Increased number may indicate a reproductive tract
infection or damage to a small capillary during sample production.
Bacteria and protozoan such as Trichomonas vaginalis are uncommon in
human semen but their presence is indicative of possible male reproductive
tract infection
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COMPUTER - ASSISTED SPERM ANALYSIS
Computer-assisted sperm analysis (CASA) is a semiautomated
technique that provides data on
Sperm density, Motility (straightline and curvilinear velocity,
linearity, average path velocity, amplitude of lateral head
displacement, flagellar beat frequency, and hyperactivation)
Advantages:
High precision
Quantitative assessment of sperm kinetics.
Disadvantages:
Expensive equipment and still requires the subjective participation
of a technician.
Hence not used for routine semen analysis
Commonly done in high volume andrology labs.
Emerging use of ICSI - diminished the role of motility assessment
in sperm selection.
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ISAS (Integrated Semen Analysis System)
SCA (Sperm Class Analyzer)
IVOS (Integrated Visual Optical System )
SQA-V (Sperm Quality Analyzer)
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ISAS (Integrated Semen Analysis System)
 ISAS is a CASA system based on image analysis.
 ISAS analyzes motility and concentration in more than 17 sperm parameters
 ISAS also do DNA fragmentation analysis
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SCA (Sperm Class Analyzer)
SCA provides fast, accurate and repeatable results.
 SCA Motility & Concentration
 SCA DNA Fragmentation
 Morphology
 SCA Vitality
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IVOS (Integrated Visual Optical System )
The IVOS is unique in that it is the only CASA system that integrates
the optical system within the unit, so that an external microscope is
not needed.
 Able to analyze sperm of multiple species (rat)
(Research institutes, IVF clinics, pharmaceutical companies,
reproductive toxicology labs, veterinary and animal breeding centres)
 A single field - analyzed in just 0.5 second.
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SQA-V (Sperm Quality Analyzer)
 Fully automated
 SQA-V semen analysis eliminates inter-operator variation.
 Electro-optics, computer algorithms and video microscopy
 Provide a precise and accurate - 75 second
The SQA-V ( 16 clinical parameters )
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Limitation of semen analysis
Clinical research has shown,
Normal semen analysis may not reflect the true fertility status of an individual.
Men with poor sperm parameters can cause spontaneous pregnancies.
Men with good sperm parameters are still subfertile
Only 50% of infertile men have recognizable causes detectable by semen analysis.
Semen analysis is only a surrogate test to measure the man’s fertility potential.
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SPERM FUNCTION ASSESSMENT
 Sperm- mucus interaction assay
 Acrosome reaction testing
 Sperm penetration assay
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SPERM-MUCUS INTERACTION/POSTCOITAL TEST
Assess cervical environment as a cause of infertility.
Cervical mucus - heterogenous fluid - cyclical changes in consistency
Postcoital test (PCT)
Conducted when the cervical mucus is thin and clear just before ovulation.
Examined 2 to 8 hours after normal intercourse.
Progressively motile sperm > 10 to 20 per HPF is designated as normal.
Abnormal test - advised to proceed with IUI.
 Inappropriate timing testing / intercourse,
 Anatomic abnormalities,
 Semen or cervical mucus antisperm antibodies,
 Abnormal sperm.
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ACROSOME REACTION
The Acrosome is a membrane-bound organelle covers the anterior 2/3 of the sperm head.
 Acrosome reaction is an important prerequisite for successful fertilization.
 ZP3
 Involves fusion of acrosomal membrane and sperm plasma membrane.
 Acrosin and Hyaluronidase – required to digest the oocyte cumulus cells and ZP
Acrosome reaction testing - not widely practiced in laboratories - research interest.
 Profound abnormalities of head morphology
 Unexplained infertility
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SPERM PENETRATION ASSAYS
The sperm penetration assay (SPA) or the hamster egg penetration assay (HEPT)
It address the functional ability.
Principle - a normal spermatozoa can bind and penetrate the oocyte membrane.
 Incubating zona-free hamster oocytes in sperm droplets for 1 to 2 hours.
 The oocytes are examined microscopically for sperm penetration.
 Penetrations are indicated by swollen sperm heads within the oocyte cytoplasm.
 Normally, 10% to 30% of ova are penetrated (WHO, 1999).
Oligozoospermic and severely teratospermic men have a higher number of defective sperm-zona
pellucida interactions, which may account for their low fertility potential in both spontaneous
and IVF pregnancies.
Sperm capacitation index (SCI) is a variant of the SPA test, assessing the mean number of
penetrations per ovum. ICSI has been recommended - SCI less than 5 instead of standard IVF
procedures.
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ADVANCED SPERM TESTING
 Antisperm antibody testing
 Electron microscopy
 Oxidative stress test
 Sperm DNA damage assay
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Antisperm Antibody Testing
AB Against sperm
IgG, IgA
 Sperm agglutinating,
 Sperm immobilizing,
 Spermotoxic.
Normally the tight Sertoli-cell junctions provide the testis with a barrier that prevents the
immune system from coming in contact with the post-meiotic germ cells.
This unique barrier can be violated,
Testicular torsion, Vasectomy,Testicular trauma, testicular surgeries
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Sperm agglutinating AB:
Agglutination of spermatozoa, which reduces
the availability of motile spermatozoa
penetrating the cervical mucus.
Sperm immobilizing AB:
Induce loss in motility of the sperm -
Characteristic “shaking” pattern in motility on
postcoital test.
Spermotoxic AB: Complement-dependent loss
in viability of spermatozoa.
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Testing of ASA
Direct ASA test detects sperm-bound immunoglobulins. (preferred)
Indirect testing detects the biologic activity of circulating ASA.
IgG-MAR (mixed antiglobulin reaction)
Sperm MAR are recommended screening tests that are economical and readily available.
Immunobead Test (IBT), which measures IgG, IgA, and IgM, may be additionally
recommended when either of the previous tests gives a positive result.
Acceptable normal values by WHO (1992) standards
Less than 10% (IgG MAR)
Less than 20% (IBT).
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Clinical implications of ASA on male infertility.
 10% of subfertile men.
 2% of fertile men.
 ASA are present in 34% to 74% of vasectomized men.
 Persist in 38% to 60% after vasectomy reversal.
Routine ASA testing is not recommended in this setting because it is of
uncertain significance and usually does not affect the decision to do a
vasectomy reversal.
Zona pellucida (ZP) test - ( IUI versus ICSI) in immunologic infertility
Inability for ZP binding, ICSI is the procedure of choice.
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ELECTRON MICROSCOPY
A viable sperm still can be defective.
Ultrastructural details of the sperm can only be seen under the electron microscope (EM).
Candidates:
Low sperm motility (<5% to 10%) with high viability & density.
Findings,
 Less intact acrosome membrane,
 More droplets attached to the acrosome membrane.
 Mitochondrial & Microtubular defects- not visible under the usual Papanicolaou smear
can be detected.
Selection of sperm for ICSI
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Reactive Oxygen Species
Excessive production (ROS) is related to abnormal semen parameters and sperm damage.
 Oxidative stress test may accurately discriminate between fertile and infertile men
 Capability is better than routine semen analysis
Currently not included in the routine evaluation of subfertile men.
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Lack of standardization of ROS analytic methods,
Lack of equipment,
Lack of normal range of ROS in semen,
Lack sufficient evidence ROS – infertility.
Chemiluminescence assay - oxidative stress status – in-vivo oxidative stress status
ROS level for healthy donors – normal semen parameters is 1.5 ×104 cpm/20 M sperm/mL.
Oxidative stress positive (>1.5 × 104 cpm/20 million sperm/mL)
Oxidative stress negative (≤1.5 × 104 cpm/20 million sperm/mL),
Regardless of their clinical diagnosis or standard semen analysis results.
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SPERM DNA DAMAGE
DNA fragmentation was initially described in 1993
Chromatin -Tightly packed.
Disulfide cross linkages between protamines.
DNA damage is multifactorial.
Protamine deficiency.
Mutations - affect DNA packaging or compaction during spermiogenesis.
Tobacco use, chemotherapy, testicular carcinoma, and other systemic cancers.
DNA damage is correlated positively with poor semen parameters, especially low
sperm concentration and low sperm motility, leukocytospermia, and oxidative
stress
Selection of sperm for ICSI
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To conclude,
Spermatogenesis –testis – FSH/LH -75days
Spermiogenesis
Stored, matured and attain motility in epididymis
Capacitation – FGT
ZP3 - Acrosome reaction, Zona reaction - fertilization - implantation.
Semen analysis
Macroscopic assessment
Microscopic assessment
Sperm function assessment
CASA but still operator dependence
Advanced sperm testing
ASA
EM
ROS
DNA damage assay
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Parameter Lower Reference Limit
Semen volume 1.5 ml
Sperm concentration 15 x 106/ml
Total sperm number 39 x106/ejaculate
Progressive motility 32 % A
Total motility 40 % A+B
Vitality (live sperms) 58 %
Sperm morphology 4 %
pH >/=7.2
Leucocyte <1 x106/ml
MAR/Immunobead test <50 %
WHO 2010
73

Thank you
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SPERMATOGENESIS, SEMEN ANALYSIS AND ANTISPERM ANTIBODIES

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