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 835.1):

 

 

Normal values for semen analysis are shown in Tables 835.1 and 835.2.

 

Test	Result
1. Volume	≥2 ml
2. pH	7.2 to 8.0
3. Sperm concentration	≥20 million/ml
4. Total sperm count per ejaculate	≥40 million
5. Morphology	≥30% sperms with normal morphology
6. Vitality	≥75% live
7. White blood cells	<1 million/ml
8. Motility within 1 hour of ejaculation
• Class A	≥25% rapidly progressive
• Class A and B	≥50% progressive
9. Mixed antiglobuiln reaction (MAR) test	<50% motile sperms with adherent particles
10. Immunobead test	<50% motile sperms with adherent particles

 

1. Total fructose (seminal vesicle marker)	≥13 μmol/ejaculate
2. Total zinc (Prostate marker)	≥2.4 μmol/ejaculate
3. Total acid phosphatase (Prostate marker)	≥200U/ejaculate
4. Total citric acid (Prostate marker)	≥52 μmol/ejaculate
5. α-glucosidase (Epididymis marker)	≥20 mU/ejaculate
6. Carnitine (Epididymis marker)	0.8-2.9 μmol/ejaculate

 

 

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.

 

 

 

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 leakproof 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. During transport to the laboratory, the specimen should be kept as close to body temperature as possible (i.e. by carrying it in an inside pocket). Ideally, the specimen should be obtained near 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 not be used for collection. Two semen specimens should be examined that are collected 2-3 weeks apart; if results are significantly different additional samples are required.

 

 

The tests that can be done on seminal fluid are shown in Box 835.2. Tests commonly done in infertility are shown in Box 835.3. The usual analysis consists of measurement of semen volume, sperm count, sperm motility, and sperm morphology.

 

Terminology in semen analysis is shown in Box 835.4.

 

 

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 are negative 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.

 

 

These tests are available only in specialized andrology laboratories. The tests are not standardized thus making interpretation difficult. If used singly, a sperm function test may not be helpful in fertility assessment. They are more predictive if used in combination.

 

 

This is the examination of the cervical mucus after coitus and 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 examinations are carried out to assess the quality of cervical mucus (elasticity and drying pattern) and to evaluate the number and motility of sperms (Box 834.1). If ≥ 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, cervicitis 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 postcoital test is 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 antibodies are suspected and male partner refuses semen analysis.

 

 

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 oocytes are enzymatically treated to remove the outer layers (that inhibit cross-species fertilization). They are then incubated with sperms and observed for penetration rate. It can be reported as (a) Number of eggs penetrated (penetration rate <15% indicates low fertility), or as (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.

 

 

This test assesses the functional integrity of the plasma membrane of the sperm by observing curling of flagella in hypo-osmotic conditions.

 

 

Computer software measures various characteristics of the spermatozoa; however, its role in predicting fertility potential is not confirmed.

 

The role of antisperm antibodies in causation of male infertility is controversial. The immunological tests done on seminal fluid include mixed antiglobulin reaction (MAR test) and immunobead test.

 

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 present bound to the head of the sperm, they will prevent the penetration of the egg by the sperm. If antibodies are bound to the tail of the sperm, they will retard motility.

 

a. SpermMAR™ test: This test can detect IgG and IgA antibodies against sperm surface in semen sample. In direct SpermMAR™ IgG test, a drop each of semen (fresh and unwashed), IgG-coated latex particles, and anti-human immunoglobulin 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 to IgG on 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 do not 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 immunoglobulin.

 

In direct 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 >50% of spermatozoa show attached latex particles, immunological problem is likely.

 

b. 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 immunobeads are counted amongst 200 motile spermatozoa. Finding of >50% spermatozoa with attached beads is abnormal.

The most important test in semen analysis for infertility is microscopic examination of the semen.

 

 

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 40× objective. Result is expressed as a percentage of motile spermatozoa observed.

 

Method: A drop of semen is placed on a glass slide, covered with a coverslip that is then ringed with petroleum jelly to prevent dehydration, and examined under 40× 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.e. crooked or curved movement), (c) non-progressive spermatozoa (movement of tails, but with no forward progress), and (d) immotile spermatozoa (no movement at all) (WHO critera). Sperms of grades (c) and (d) are considered to be poorly motile (asthenospermia). Normally, ≥ 25% of sperms show rapid progressive motility, or ≥ 50% of sperms show rapid progressive and slow progressive motility.

 

If the proportion of motile spermatozoa is < 50%, then proportion of viable sperms should be determined by examining an eosin preparation.

 

 

Principle: A cell with intact cell membrane (a vital or viable cell) will not take up 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 be stained pink-red (Figure 832.1). Another stain (e.g. nigrosin) may  be used to stain the background material. The test is performed if motility is abnormal.

 

 

 

 

Seventy-five percent or more of sperms are normally live or viable.

 

 

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).

 

 

 

 

A smear is prepared by spreading a drop of seminal fluid on a glass slide, stained, and percentages of normal and abnormal forms of spermatozoa are counted. The staining techniques used are Papanicolaou, eosinnigrosin, hematoxylin-eosin, and Rose Bengal-toluidine blue stain. Atleast 200 spermatozoa should be counted under oil immersion. Percentages of normal and abnormal spermatozoa should be recorded.

 

Normal morphology: A spermatozoon consists of three main components: head, neck, and tail. Tail is further subdivided into midpiece, main (principle) piece, and end piece (Figure 832.2 and Box 832.1).

 

 

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 acrosomal cap. Acrosomal cap is a flattened membranebound 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.

 

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, > 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).

 

 

Abnormal morphology (Figure 832.3): WHO morphological classification of human spermatozoa (1999) is given below:

 

 

 

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/ml indicate presence of infection. Presence of large number of immature sperm cells indicates spermatogenesis dysfunction at the testicular level.

Biochemical markers (Table 831.1) can be measured in semen to test the secretions of accessory structures. These include fructose (seminal vesicles), zinc, citric acid or acid phosphatase (prostate), and α-glucosidase or carnitine (epididymis).

 

1. Total fructose (seminal vesicle marker)	≥13 μmol/ejaculate
2. Total zinc (Prostate marker)	≥2.4 μmol/ejaculate
3. Total acid phosphatase (Prostate marker)	≥200U/ejaculate
4. Total citric acid (Prostate marker)	≥52 μmol/ejaculate
5. α-glucosidase (Epididymis marker)	≥20 mU/ejaculate
6. Carnitine (Epididymis marker)	0.8-2.9 μmol/ejaculate

 

 

Resorcinol method is used for detection of fructose. In this test, 5 ml of resorcinol reagent (50 mg resorcinol dissolved in 33 ml concentrated hydrochloric acid; dilute up to 100 ml with distilled water) is added to 0.5 ml of seminal fluid. The mixture is 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 a lack of seminal vesicles. In a case of azoospermia, if fructose is absent, it is due to the obstruction of ejaculatory ducts or absence of vas deferens, and if present, azoospermia is due to failure of testes to produce sperm.

Examination is carried out after liquefaction of semen that occurs usually within 20-30 minutes of ejaculation.

 

 

Normal semen is viscous and opaque gray-white in appearance. After prolonged abstinence, it appears slightly yellow.

 

 

Immediately following ejaculation, normal semen is thick and viscous. It becomes liquefied within 30 minutes by the action of proteolytic enzymes secreted by prostate. If liquefaction does not occur within 60 minutes, it is abnormal. The viscosity of the sample is assessed by filling a pipette with semen and allowing it to 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; it results from infection of seminal vesicles or prostate.

 

 

Volume of 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.

 

 

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 (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).