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‘Semen (or seminal fluid) is a fluid that is emitted from the male genital tract and contains sperms that are capable of fertilizing female ova. Structures involved in production of semen are (Box 182: Testes: Male gametes or spermatozoa (sperms) are produced by testes; constitute 2-5% of semen volume. + Epididymis: After emerging from the testes, sperms are stored in the epididymis where they mature; potassium, sodium, and glycerylphosphoryicholine (an energy source for sperms) are secreted by epididymis. + Vas deferens: Sperms travel through the vas deferens totheampulla which is another storage area. Ampulla secretes ergothioneine (a yellowish fluid that reduces chemicals) and fructose (source of nutrition for sperms). + Seminal vesicles: During ejaculation, nutritive and lubricating fluids secreted by seminal vesicles and pprostateare added. Fluid secreted by seminal vesicles consists of fructose (energy source for sperms), amino acids, citric acid, phosphorous, potassium, and prostaglandins, Seminal vesicles contribute 50% to semen volume. Prostate: Prostatic secretions comprise about 40% of semen volume and consist of citric acid, acid phosphatase, calcium, sodium, zinc, potassium, proteolytic enzymes, and fibrolysin. * Bulbourethral glands of Cowper secrete mucus. | Box 15.1: Contributions to semen volume + Testes and epididymis: 10% + Seminal vesictes: 50% + Prostate: 40% + Cowper's glands: Small volume ‘Normal values forsemen analysis are shown in Tables 18. and 152. INDICATIONS FOR SEMEN ANALYSIS Availability of semen for examination allows direct examination of male germ cells that is not possible with female germ cells. Semen analysis requires skill and should preferably be done in a specialized andrology laboratory. 1. Investigation of infertility: Semen analysis is the first ‘step in the investigation of infertility. About 30% cases, of infertility are due to problem with males. 2. To check the effectiveness of vasectomy by confir- ming absence of sperm. 3, To support or disprove a denial of patemity on the grounds of sterility. 4, To examine vaginal secretions or clothing stains for the presence of semen in medicolegal cases. 5. For selection of donors for artificial insemination. 6, Forselection of assisted reproductive technology,eg. in vitro fertilization, gamete intrafallopian transfer technique. COLLECTION OF SEMEN FOR INVESTIGATION OF INFERTILITY Semen specimen is collected after about 3 days of sexual abstinence. Longer period of abstinence reduces motility of sperms. If the period of abstinence is shorter than 3 days, sperm count is lower. The sample is obtained by ‘masturbation, collected in a clean, dry, sterile, and leak- proof wide-mouthed plastic container, and brought to the laboratory within 1 hour of collection. The entire ejaculate is collected, as the first portion is the most concentrated and contains the highest number of sperms. Essentials of Clinical Pathology rable 151: Normal values of semen analysis (World Heath Organization, 1698) 1 ee | 22 mi Volume pH : rm concentration ‘Total sperm count per ejaculate Morphology Vitality ‘White blood cells Motility within 1 hour of ejaculation = Class A. + Class A and B ‘Mixed antiglobuiln reaction (MAR) test immunobead test ‘Table 15.2: Biochemical variables of 1. Total fructose (seminal vesicle marker) 2. Total zine (Prostate marker) 3, Total acid phosphatase (Prostate marker) 4, otal cite acid (Prostate marker) 5. a-glucosidase (Epididymis marker) 6. Camitine (Epididymis marker) During transport to the laboratory, the specimen should bbe kept as close to body temperature as possible (i.e. by carrying it in an inside pocket). Ideally, the specimen should be obtained rear the testing site in an adjoining room. Condom collection is not recommended as it contains spermicidal agent. Ejaculation after coitus interruptus leads to the loss of the first portion of the ejaculate that is most concentrated; therefore this method should net be used for collection. Two semen specimens should be examined that are collected 23 weeks apart; if results are significantly different additional samples, are required. EXAMINATION OF SEMINAL FLUID ‘The tests that can be done on seminal fluid are shown in Box 15.2. Tests commonly done in infertility are shown in Box 153. The usual analysis consists of measurement of semen volume, sperm count, sperm motility, and ‘sperm morphology. Terminology in semen analysis is shown in Box 15.4. 721 81 220 milion’! 240 milion 320% sperms with normal morphology: 275% live ‘<1 million’! 225% rapidly progressive on progressive 0% mote aperme with adherent particles * ‘250% motile sperms with adherent particles: ‘semen analysis (World Helath Organization, 1992) 213 ymoViejaculate 224 ymol/ejacuiate 2200Ulejaculate 252 jmoliejaculate +220 mUlejaculate 08-29 ymoliejaculate Box 15.2: Tests done on seminal fluid | ae cue pair reat | [ee ad Rc secon! Pete ta recs coeenn Serer ssa tae Biochemical analysis: Fructose, zinc, acid phosphatase, camitine. |¢ Sperm function tests: Postcoital test, cervical mucus, penetration test, Hamster egg penetration assay, hypo- ‘osmotic swelling of agela, and computer-assisted semen Box 15.3: Semen analysis for initial investigation of infertility + Volume + pH sscopic examination for ()) percentage of motile ‘spermatozoa, (1) sperm count, and (i) sperm morphology (ld to moderate: 5-20 milion/ml; severe: <5 milion/m!) ‘Azpospermia: Absence of sperms in seminal fluid i ‘Aspermia: Absence of ejaculate | ‘Asthenozoospermia: Reduced sperm motility; <50% of ‘sperms showing class (a) and ciats (b) type of motity OR <25% sperms showing class (a) type of motility, + Teratozoospermia: Spermatozoa with reduced proportion fren morpelogy (or nereased proportion efabremmal rm) | t Leukocytospermia: >1 million white blood cells/ml of | + Oligoasthencteratezoospermia: All sperm variables, are abnormal + Necrozoospermia: All sperms are non-motle or non-viable: Physical Examination Examination is carried out after liquefaction of semen that occurs usually within 20-30 minutes of ejaculation. 1, Visual appearance: Normal semen is viscous and ‘opaque gray-white in appearance. After prolonged abstinence, it appears slightly yellow. ‘Viscosity: Immediately following ejaculation, normal semen is thick and viscous. It becomes liquefied within 30 minutes by the action of proteolytic enzymes secreted by prostate. Ifliquefaction does not ‘occur within 60 minutes, itis abnormal. The viscosity of the sample is assessed by filling a pipette with semen and allowing itto flow back into the container. ‘Normal semen will fall drop by drop. If droplets form ‘threads’ more than 2 cm long, then viscosity is increased. Increased semen viscosity affects sperm ‘motility and leads to poor invasion of cervical mucus; itresults from infection of seminal vesicles or prostate. 3. Volume: Volumeof ejaculated semen sample should normally be > 2 ml. It is measured after the sample has liquefied. Volume < 2.0 ml is abnormal, and is associated with low sperm count. 4. pH: A drop of liquefied semen is spread on pH paper (of pH range 6.4-8.0) and pH is recorded after 30 seconds. Normal pH is 7.2 to 8.0 after 1 hour of ejaculation. The portion of semen contributed by ‘seminal vesicles is basic, while portion from prostate is acidic. Low pH (< 7.0) with absence of sperms Semen Andiyeie? FE (azoospermia) suggests obstruction of ejaculatory ducts or absence of vas deferens. Low pH is usually associated with low semen volume (as most of the ‘volume is supplied by seminal vesicles). Microscopic Examination ‘The most important test in semen analysis for infertility {s microscopic examination of the semen. ‘Sperm Motility ‘The first laboratory assessment of sperm function in a ‘wet preparation is sperm motility (ability of the sperms to move). Sperm motility is essential for penetration of cervical mucus, traveling through the fallopian tube, and ‘penetrating the ovum. Only those sperms having rapidly progressive motility are capable of penetrating ovum and fertilizing it. Principle: All motile and non-motile sperms are counted in randomly chosen fields in a wet preparation under 40x objective. Result is expressed as a percentage of ‘motile spermatozoa observed. Method: A drop of semen is’placed on-a glais slide, covered with a coverslip that is then ringed with petroleum jelly to prevent dehydration, arid examined ‘under 40x objective. Atleast 200 spermatozoa are counted in several different microscopic fields. Result is expressed as a percentage of (a) rapidly progressive spermatozoa (moving fast forward in a straight line), (b) slowly progressive spermatozoa (slow linear or non-linear, i. crooked or curved movement), (c) non-progressive ‘spermatozoa (movernent of tails, but with no forward progress), and (d) immotile spermatozoa (no movement at all) (WHO critera). Sperms of grades (c) and (A) are considered to be poorly motile (asthenospermia). Normally, 2 25% of sperms show rapid progressive ‘motility, or250% of sperms show rapid progressive and slow progressive motility. If the proportion of motile spermatozoa is <50%, then proportion of viable spernis should be determined by ‘examining an eosin preparation. Sperm Viebilty or Vitality Principle: A cell with intact cell membrane (a vital or viable cell) will not take tip the eosin Y and will not be stained, while a non-viable’or dead cell ‘will have ‘damaged cell membrane, will take up the dye, and will Essentials of Clinical Pathology Fig. 15.1: Eosin-rigrosin stain. Dead sperms are stained pink-red, while live sperms are stained white be stained pink-red (Fig. 15.1). Another stain (e.g. nigrosin) may be used to stain the background material, The testis performed if motility is abnormal, Method 1. Mixone drop of semen with 1 drop of eosin-nigrosin solution and incubate for 30 seconds. 2. A'smear is made froma drop placed on a glass slide. 3. The smear is airadried and examined under oil- immersion objective. White sperms are classified as live or viable, and red sperms are classified as dead ‘ornon-viable, Atleast 200 spermatozoa are examined. 4, The result is expressed as a proportion of viable sperms against non-viable as an integer percentage. Seventy-five percent or more of sperms are normally live or viable. ‘Sperm Count Principle: The sperm count is done after liquefaction in a counting chamber following dilution and the total ‘number of spermatozoa is reported in millions/ml (106/ ml). Method 1. Semen is diluted 1:20 with sodium bicarbonate- formalin diluting fluid (Take 1 ml liquefied semen in ‘a graduated tube and fill with diluting fluid to 20 ml mark. Mix well) 2. A coverslip is placed over the improved Neubauer counting chamber and the counting chamber is filled with the well-mixed diluted semen sample using a Pasteur pipette. The chamber is then placed in a humid box for 10-15 minutes for toz08 to settle, 3, Thechamberis placed on the microscope stage. Using the 20x or 40x objective and iris diaphragm lowered sufficiently to give sufficient contrast, number of spermatozoa is counted in 4 large comer:squares. Spermatozoa whose heads are touching left and upper lines of the square should be considered as ‘belonging’ to that square. 4. Sperm count per ml is calculated as follows: ‘Sperms counted xcontction factor fordilution, Numberof \%. jVolumeof==* squarescounted , lequare, yy ‘Sperms counted » 20 ro aa oe Spermcount = 1000 = Sperms counted 50,000 5. Normal sperm count is 220 million/ml (Le. 2 20 x 10/ml). Sperm count < 20 million/m may be associated with infertility in males, ‘Sperm Morphology ‘A smear is prepared by spreading a drop of seminal fluid con a glass slide, stained, and percentages of normal and abnormal forms of spermatozoa are counted. The staining techniques used are Papanicolaou, eosin- nigrosin, hematoxylin-eosin, and Rose Bengal-toluidine blue stain. Atleast 200 spermatozoa shoitld be counted under oil immersion. Percentages of riormial and ‘abnormal spermatozoa should be recorded. Normal. morphology: A spermatozoon consists of three main components: head, neck, and tail. Tal is further subdivided into midpiece, main (principle) piece, and ‘end piece (Fig. 15.2 and Box 155). Head is pear-shaped. Most of the head is occupied by the nucleus which has condensed chromatin and few ‘areas of dispersed chromatin (called nuclear vacuoles). ‘The anterior 2/3rds of the nucleus is surrounded by ‘crosomal cap. Acrosomal cap isa flattened membrane- bound vesicle containing glycoproteins and’ enzymes. These enzymes are required for separation of cells of corona radiata and dissolution of zona pellucida of ovum during fertilization. | End piece Fig. 15.2: Morphology of spermatozoa Neck is a very short segment that connects the head and the tail. Centriole in the neck gives rise to axoneme of the flagellum. Axoneme consists of 20 microtubules (arranged as a central pair surrounded by 9 peripheral doublets) and is surrounded by condensed fibrous rings. ‘Middle piece is the first part of the tail and consists of central axoneme surrounded by coarse longitudinal fibers. These are surrounded by elongated mitochondria that provide energy for movement of tail. Principle or main piece constitutes most of the tail and is composed of axoneme that is surrounded by 9 coarse fibers. This central core is surrounded by many circularly arranged fibrous ribs. Endpiece is the short tapering part composed of only axoneme. ‘Normally, 2:30% of spermatozoa should show normal morphology (WHO, 1999). The defects in morphology that are associated with infertility in males include defective mid-piece (causes reduced motility), an incomplete or absent acrosome (causes inability to penetrate the ovum), and giant head (defective DNA. condensation). Semen Analysis flag [hen $88: Norma! orvmonnelega | |+ Total length of sperm; About 60-1. | Head: = Length: 3-5 5. = Width: 23 = Thickness: 1.5 Neo: Length: 0.3 Middle piece: = Length: 36 . = Wath: 1.0 Principal piece: =. Length: 40-50 = Width: 05 End piece: 4-6 “Abnormal morphology (Fig. 153): WHO morphological classification of human spermatozoa (1999) is given below: 1. Normal sperm 2 Defects in head: + Large heads ‘Small heads H Tapered heads Pyriform heads Round heads “Amorphous heads ‘Vacuolated heads (> 20% of the head area occu- pied by vacuoles) + Small acrosomes (occupying < 40% of head area) ‘= Double heads 3. Defects in neck: ‘+ Bent neck and tail forming an angle >90° to the long axis of head 4. Defects in middle piece: «Asymmetric insertion of midpiece into head Thick or irregular midpiece + Abnormally thin midpiece 5. Defects in tail: Bent tails Short tails 3 Coiled tails Irregular tails Multiple tails Tails with irregular width Pin heads: Not to be counted . Cytoplasmicdroplets ‘+ >'1/3ed the size of the sperm head 8. Precursor cells: Considered abnormal 2 Essentials of Clinical Pathology 1 \—rerosome 2 Middle ace" Principal piece End piece Fig. 18. hhead, (11) Pin Round Cells Round cells on microscopic examination may be white blood cells or immature sperm cells. Special stain (peroxidase or Papanicolaou) is required to differentiate between them. White blood cells >1 million/m indicate presence of infection. Presence of large number of immature sperm cells indicates spermatogenesis, dysfunction at the testicular level. Immunologic Analysis Antisperm Antibodies ‘The role of antisperm antibodies in causation of male infertility is controversial. The immunological tests done fon seminal fluid include mixed antiglobulin reaction (MAR test) and immunobead test. i The antibodies against sperms immobilize or kill them, thus preventing their passage through the cervix to the ovum. The antibodies can be tested in the serum, seminal fluid, or cervical mucus. If the antibodies are ppresentbound to the head of the sperm, they will prevent the penetration of the egg by the sperm. Ifantibodies are bound to the tail of the sperm, they will retard motility. (12) Round head without acrosome and thick midpiece, (13) Coiled tall, and (14) Double tail ‘Abnormal morphological sperm forms: (1) Normal sperm, (2) Large head, (3) Small head, (4) Tapered hea Pynitorm head, (8) Round head, (7) Amorphous head, (8) Vacuoles in head, (9) Round head without acrosome, ( ) Double ‘a. SpermMAR™ test: This test can detect IgG and IgA antibodies against sperm surface in semen sample. Indirect SpermMAR™ IgG test,a drop each of semen (fresh and unwashed), IgG-coated latex particles, and anti-human imm« in are mixed together on a glass slide. At least 200 motile spermatozoa are examined. If the spermatozoa have antibodies on their surface, antihuman immunoglobulin will bind IgG-coated latex particles tolgGon the surface of the spermatozoa; this will cause attachment of latex particles to spermatozoa, and motile, swimming sperms with attached particles will be seen. If the spermatozoa donot have antibodies on their surface, they will be seen swimming without attached particles; the latex particles will show clumping due to binding of their IgG to antihuman immuno- globuli Indirect SpermMAR™ Iga test,a drop each of fresh unwashed semen and of IgA-coated latex particles, are mixed on a glass slide. The latex particles will bind to spermatozoa if spermatozoa are coated with IgA antibodies. In indirect SpermMAR™ tests, fluid without ‘spermatozoa (e.g, serum) is tested for the presence of antisperm antibodies. First, antibodies are bound to donor spermatozoa which are.then mixed with the fluid to be analyzed. These antibodies are then detected as described above for direct tests. ‘Atleast 200 motile spermatozoa should be counted. If 550% of spermatozoa show attached latex particles, immunological problem is likely. . Immunobead test: Antibodies bound to the surface of the spermatozoa can be detected by antibodies attached to immunobeads (plastic particles with attached anti-human immunoglobulin that may be either IgG, IgA,or IgM). Percentage of motile spermatozoa with attached two or more immuno- ‘beads are counted amongst 200 motile spermatozoa. Finding of >50% spermatozoa with attached beads is abnormal. Biochemical Analysis of Semen Biochemical markers (Table 15.2) can be measured in semen to test the secretions of accessory structures. These include fructose (seminal vesicles), zine, citric acid or acid phosphatase (prostate), and a-glucosidase or carnitine (epididymis). Test for Fructose Resorcinol method is used for detection of fructose. In this test, 5 ml of resorcinol reagent (50 mg resorcinol dissolved in33 ml concentrated hydrochloric acid;dilute up to 100 ml with distilled water) is added to 0.5 ml of seminal fluid. The mixtures heated and brought to boil, If fructose is present,a red-colored precipitate is formed within 30 seconds, ‘Absence of fructose indicates obstruction proximal to seminal vesicles (obstructed or absent vas deferens) ‘or lack of seminal vesicles. Ina case of azoospermia, if fructose is absent, it is due to the obstruction of ejaculatory ducts or absence of vas deferens, and if resent, azoospermiais due to failure of testes to produce sperm. Sperm Function Tests or Functional Assays ‘These tests are available only in specialized andrology laboratories. The tests are not standardized thus making interpretation dificult. If used singly, a sperm function test may not be helpful in fertility assessment. They are more predictive if used in combination, Semen Analysis, ff Posteoital (Sims-Huhner) Test Thisis the examination ofthe cervical mucus after coitus land assesses the ability of the sperm to penetrate the cervical mucus. The quality of the cervical mucus varies during the menstrual cycle, becoming more abundant and fluid at the time of ovulation (due to effect of estrogen); this facilitates penetration of the mucus by the spermatozoa. Progesterone in the secretory phase increases viscosity of the mucus. Therefore cervical mucus testing is scheduled just before ovulation (determined by basal body temperature records or follicular sizing by ultrasonography). Postcoital test is the traditional method to detect the cervical factor in infertility. Cervical mucus is aspirated with a syringe shortly before the expected time of ovulation and 2-12 hours after intercourse.Gross and microscopic examina- tions are carried out to assess the quality of cervical ‘mucus (elasticity and drying pattern) and to evaluate the ‘number and motility of sperms (Box 15). 1f2 10 motile sperms are observed the test is considered as normal. ‘An abnormal test may result from: (a) poor quality of cervical mucus due to wrong judgment of ovulation, cervictis or treatment with antioestrogens (e.g. Clomid), and (b) absence of motile sperms due to ineffective technique of coitus, lack of ejaculation, poor semen quality, use of coital lubricants that damage the sperm, or presence of antisperm antibodies. Antisperm antibodies cause immotile sperms, or agglutination or clumping of sperms; they may be present in either partner. If cervical factor is present, intrauterine insemination is the popular treatment. The value of the ppostcoital testis disputed in the medical literature, This test can be carried out if semen analysis is ‘normal,and the female partner is ovulating and fallopian tubes are not blocked. It is also done if antisperm Box 15.8: Interpretation of postroltal test * Normal: Sperms are normal in amount and moving forward | in the mucus; mucus stretches atleast 2 inches (5 cm)| | and dries in a fern-ike manner. + Abnormat Absence of sperms or large number of sperms! ‘are dead or sperms are clumped; cervical mucus cannet ‘stretch 2 inches (5 em) or does not dry in a, fer-ike BL] Essentials of Clinical Pathology antibodies are suspected and male partner refuses semen analysis, Cervical Mucus Penetration Test In,this test, greatest distance traveled by the sperm in seminal fluid placed and incubated in a capillary tube containing bovine mucus is measured. Majority of fertile men show score >30 mm, while most infertile men show scores <20 mm. Hamster Egg Penetration Assay Hamster oocytes are enzymatically treated to remove the ‘cuter layers (that inhibit cross-species fertilization). They are then incubated with sperms and observed for Penetration rate. Itcan be reported as (a) Number of eggs Penetrated (penetration rate <15% indicates low fertility), ras (b) Number of sperm penetrations per egg (Normal >5). This test detects sperm motility, binding to oocyte, and penetration of oocyte. There is a high incidence of false-negative results, Hypo-osmotic Swelling of Flagella This test assesses the functional integrity of the plasma membrane of the sperm by observing curling of flagella in hypo-osmotic conditions. Computer-assisted Semen Analysis Computer software measures various characteristics of the spermatozoa; however, its role in predicting fertility potential is not confirmed. EXAMINATION FOR THE PRESENCE OF SEMEN IN MEDICOLEGAL CASES This includes examination of material obtained from vagina, stains from clothing, skin, hair, or other body parts for semen. This is carried out in cases of alleged Tape or sexual assault, Collection of Sample ‘+ Vagina: Direct aspiration or saline lavage * Clothing: When scanned with ultraviolet light, semen Produces green white fluorescence. A small piece (1 1) of clothing from stained portion is soaked in 1-2 mlof physiologic saline for | hour. A similar piece of clothing distant from the stain is also soaked in saline asa control Tests |. Microscopic examination for sperms: Presence of motile sperms in vaginal fluid indicates interval of <8 hours. Smears prepared from collected samples arestained and examined for the presence of sperms. 2. Acid phosphatase: Acid phosphatase is determined on vaginal or clothing samples. Due to the high level of ‘acid phosphatase in semen, its presence indicates recent sexual intercourse. Level of 250 U/sample is considered as positive evidence of semen. 3. Determination of blood group substances: When semen is positively identified in vaginal fluid or other sample, test can be carried out for the presence of blood group substances in the same sample. The ‘secretor’ individuals (80% individuals are secretors) will secrete the blood group substances in body fluids, including semen. 4. Florence test: This test detects the presence of choline found in high concentration in semen. To several drops of sample, add equal volume of reagent (iodine 2.54 g, potassium iodide 1.65 g distilled water 30 ml); in positive test rhombic or needle-like crystals of periodide of choline form. False-positive tests can occur due tohigh choline content of some otherbody fluids. EXAMINATION OF SEMEN TO CHECK THE EFFECTIVENESS OF VASECTOMY The aim of post-vasectomy semen analysis is to detect the presence or absence of spermatozoa. The routine follow-up consists of semen analysis starting 12 weeks (or 15 ejaculations) after surgery. If two successive semen samples arenegative for sperms, the semen is considered as free of sperm. A follow-up semen examination at 6 months is advocated by some to rule out spontaneous reconnection. BIBLIOGRAPHY 1. Hirsh A. Male subfertiity. BMJ 2003;327:669-72 2. Phadke AM. Clinical Atlas of Spermn Morphology. New Delht Jaypee Brothers Medical Publishers (P) Lid. 2007. 3. World Health Organization, Laboratory Manual forthe Examination of Human Semgn and Sperm-Cervical Mucus interactions 3rd edition. Cambridge: Cambridge University Press, 1992. 4. World Health Organization. WHO laboratory manual for the examination of human semen and sperm ‘cervical mucus interaction. 4th edition. UK: ‘New York NY: Published on behalfof the World Health Organization (by] Cambridge University Press; 1999,

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