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SEMEN ANALYSIS Dr. Santanu Ghosh, MD (Psy), Ph.D (Psyc) Associate Professor, Psychiatry, Tripura Medical College, Agartala, Tripura
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5 Introduction 4 3 2 1 Indication of semen analysis Semen Collection Semen Analysis Computer assisted semen analysis Layout of Presentation
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Introduction A semen analysis measures the amount of semen a man produces and determines the number and quality of sperm in the semen sample. A semen analysis is usually on e of the first test done to exclude paternal cause of infertility A problem with the semen or sperm affects more than one- third of the couples who are unable to have children (infertile)
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The Ejaculate
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C B A Part of Investigation in Primary and secondary Infertility Test for vasectomy success (usually done 6 weeks after the vasectomy) Demand from court of law in legal disputes Indication of Semen Analysis Since semen samples may vary from day to day, 2 or 3 samples may be evaluated within a 3-6 month period for accurate testing
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Human sperm cell is about 70 µm long. The head size: 4-5µm Nucleus - contains the 23 chromosomes. Acrosome Mid-piece: 4-5µm The energy for motility is generated. Tail: 55µm Motility -Propagated along the tail.
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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.
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PRECAUTIONS 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. Collect the entire sample -70% of sperms is in the first part of the ejaculate. Other methods of collection Coitus interrupts Condom collection - Polyurethane - Latex
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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 Vibratory stimulation - spinal cord injury. Rectal probe electro - stimulation induces ejaculation by stimulation of the efferent fibers of the hypo gastric plexus.
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LABELLING OF SAMPLE Patient name Age Clinic or Doctor name Laboratory analysis form: The period of abstinence (in days). Date &Time of collection. Mode of collection. Complete or incomplete. The time interval from collection to analysis.
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TIMING OF ANALYSIS 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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Terminologies in SA (WHO) Leucospermia - Leucocyte present in semen, > 1 M/ml
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Normospermia Aspermia Hypospermia Hyperspermia Azoospermia Oligospermia Polyzoospermia Asthenozoospermia Teratozoospermia Leukospermia Hematospermia Necrozoospermia Normal semen volume No semen volume Semen volume < 1.5 ml Semen volume > 6.0 ml No spermatozoa in semen Sperm concentration <15 M/ml High sperm concentration, >200M/ml <40% grade (A&B) or < 32 PR% <4% spermatozoa - Leukocytes present in semen, >1M/ml Red blood cell present in semen “dead” sperm
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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 20x magnification Sperm density = Sum of 5 squares x 106/ml
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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 Seminal vesicles and prostate, 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) Ejaculatory duct obstruction. Macroscopic Examination
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Macroscopic Examination…cont WHO criteria 2010 Description Whitish to grey opalescent Appearance Normal: Yellow (urine, jaundice) Pink/Reddish/Brown (RBCs) Liquefaction Normal: 15–30 minutes after collection >60 min Lack of prostatic protease, maybe sign of prostatic infection Viscosity Normal Smooth and watery Abnormal thick with long threads.
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Semen is ejaculated in liquid state. It gets coagulated due to enzyme PROTEIN KINASE from seminal vesicles. Absence of coagulation indicates CONGENITAL ABSENCE OF VAS DEFERENS,SEMINAL VESICLES OR OBSTRUCTION OF THE EJACULATORY DUCT LIQUEFACTION AT ROOM TEMPERATURE ,NORMAL SEMEN GETS LIQUEFIED WITHIN 30 MINUTES AFTER COLLECTION. Presence of MUCOUS STREAKS indicate incomplete liquefaction. Sometimes the sample may not liquefy. In this situation a treatment with PLASMIN 0.35 – 0.50 UNITS/ ML or CHYMOTRYPSIN 150 USP / ML may be needed to make the sample fit for analysis. Incomplete liquefaction is indicative of dysfunctional accessory reproductive organs like prostate which leads to decreased production of prostatic enzymes.
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FRUCTOLYSIS Fructose is main sugar present in seminal plasma & is imp. nutrient for the sperms. The quality of semen can be assessed by measuring the rate of utilization of fructose. FRUCTOLYTIC INDEX is the amount of fructose used or lactic acid formed by spermatozoa per hour at 37 deg. The semen sample should be well buffered otherwise the fructolysis will stop at certain stage & result will be erroneous. The normal fructose value is 13 mol or more per ejaculate. In case of azoospermia caused by congenital absence of vas deferens , low fructose level may indicate an assoc. dysgenesis of seminal vesicles. Fructose determination is also useful in rare cases of ejaculatory duct obstruction. There is positive correlation between rate of anaerobic fructolysis & deg. of motility.
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MICROSCOPIC ASSESSMENT OF SEMEN Sperm agglutination Count and concentration Motility Morphology Viability Non sperm cells
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SPERM AGGLUTINATION Wet smear Sperm form clumps within semen Sperm-to-non sperm elements (nonspecific agglutination)- accessory gland infection. Sperm-to-sperm agglutination (site-specific agglutination) – anti sperm 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. 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—Non progressive 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, Varicocele. ASA - peculiar shaking pattern – preventing penetration through cervical mucus. > 10% to 15% of clumping of spermatozoa is indicative of antisperm antibodies
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HABITUAL FACTORS AFFECTING SPERM DENSITY / MOTILITY High intake of soya – decrease sperm density. High consumption of tobacco – decrease sperm density / motility. Consumption of cocaine / Marijuana – decrease sperm motility. Vaginal lubricants – decrease sperm motility. Alcoholism – affects all semen parameters.
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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 hypo osmotic 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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NON SPERM CELLS Leukocytes: normally (1-4/HPF) Leukocytospermia as levels above 1 × 106 WBC/mL - infection 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 SEMEN ANALYSIS (CASA)
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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. Computer-assisted sperm analysis (CASA) It is a semiautomated technique that provides data on Sperm density, Motility (straight line and curvilinear velocity, linearity, average path velocity, amplitude of lateral head displacement, flagellar beat frequency, and hyper activation)
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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 subfertile 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 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. Unexplained infertility / IVF failure 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 – negative testing 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 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. Sperm MAR(mixed antiglobulin reaction ) are recommended screening tests that are economical and readily available. Immunobead Test (IBT), which measures IgG, IgA, and IgM, may be additionally recommended when the previous tests gives a positive result. Acceptable normal values by WHO (1992) standards Less than 10% (IgG MAR) Less than 20% (IBT). Less than 50% WHO 2010
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Clinical implications of ASA on male infertility. 10% of sub fertile men. 2% of fertile men. ASA are present in 34% to 74% of vasectomized men. Persist in 38% to 60% after vasectomy reversal. Does not affect the decision to do a vasectomy reversal. Routine testing is not recommended (IVF 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. Ultra structural 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 & Micro tubular defects- not visible under the usual Papanicolaou smear can be detected. Selection of sperm for ICSI
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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. Selection of sperm for ICSI
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PRE – EJACULATORY FRACTION 1. Example Text 2. Example Text 3. Example Text 4. Example Text The Ejaculate Normal semen is an admixture of spermatozoa suspended in secretions (SEMINAL PLASMA ) from the glandular tissue of the male genital system. The ejaculate can be divided into four fractions: 1) PRE – EJACULATORY FRACTION : It is clear secretion of COWPER’S or LITTER’S GLANDS & contains proteins with moderately viscous consistency, which may possibly serve to neutralize residues of urine . 2)PRELIMNARY FRACTION : This originates from the prostrate gland. It gives SEMEN it’s characteristic ODOUR. It contain enzymes which liquefies the spermatozoa coagulum. 3)MAIN FRACTION : It originates from the SEMINAL VESICLES, TESTES, EPIDIDYMIS & partially from the prostate gland. The preliminary fraction & the main fraction contain majority of spermatozoa. 4)TERMINAL FRACTION : Is formed by secretions of seminal vesicles & is entirely gelatinous in consistency ,with large no. of immotile spermatozoa.
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