Semen Analysis: Laboratory examination of Semen

Preparation

• Specimen collection should be done in a private room near the laboratory to minimize exposure of the semen to fluctuations in temperature and to control the time between collection and examination.

• The sample must be obtained after 3 days and a maximum of 7 days of sexual abstinence. If additional samples are needed, the number of days of sexual abstinence should be as constant as possible at for each visit.

• The client is be given a clear written and spoken directions concerning the collection of the sample. These directions should emphasize that the semen sample needs to be complete and that the man should report any loss of any fraction of the sample.

• The following client information must be included on the report form: the man’s name, birth date and personal code number, the period of abstinence, the date and time of collection, the completeness of the sample, any difficulties in collecting the sample, and the interval between collection and the start of the semen examination.

Semen collection is obtained by masturbation and ejaculated into a clean, wide-mouthed container made of glass or plastic, from a batch that has been confirmed to be non-toxic for spermatozoa.

Specimen container should be kept at an ambient temperature, between 20 °C and 37 °C, to avoid large changes in temperature that may gradually affect the spermatozoa after they are ejaculated into it.

The specimen container t must be labelled with the man’s name and identification number, and the date and time of collection.

The specimen container is placed on the bench or in an incubator (37 °C) for liquefaction.

Include in the report if the semen sample is incomplete, especially if the first, sperm-rich fraction ismissing. If the sample is incomplete, a second sample must be collected, again after an abstinence period of 3–7 days.

Specimen collection: How semen is collected for assisted reproduction

This is done as for diagnostic collection but the specimen containers, pipette tips and pipettes for mixing should be sterile.

Specimen collection: Sterile semen collection for microbiological analysis

For this case microbiological contamination from non-semen sources (e.g. commensal organisms from the skin) must be avoided completely. The specimen containers, pipette tips and pipettes for mixing must be sterile.

The man should;

• First pass out rine.
•  Wash his hands and penis with soap, to reduce the risk of contamination of the specimen with commensal organisms from the skin.
• Rinse away the soap.
• Dry his hands and penis with a fresh disposable towel.
• Ejaculate into a sterile container.

Note:

The time between collection of the semen sample and the start of the investigation by the microbiological laboratory should not go beyond 3 hours.

Collection of semen sample at home

A semen sample can be collected at home in exceptional situations, like in demonstrated inability to produce a sample by masturbation in the facility or the lack of adequate facilities near the laboratory.

The client should be given clear written and spoken directions concerning the collection and transport of the semen sample. These should emphasize that the semen sample has to be complete, thus, all the ejaculate is collected, including the first, sperm-rich portion, and that the man should report any loss of any fraction of the sample.

It should be included in the report if the sample is incomplete.

The client should be given a pre-weighed container, labelled with name and identification number.

The client should note down the time of semen production and deliver the sample to the laboratory within 1 hour of collection.

During transport to the laboratory, the sample must be kept between 20 °C and 37 °C.

The report should include place of sample collection

Collection of semen sample by condom

A semen sample can be collected in a condom during sexual intercourse only in exceptional circumstances, such as a inability to produce a sample by masturbation.

Only special non-toxic condoms designed for semen collection must be used.

The client should be given information from the manufacturer on directions for condom use, close it, and send or transport it to the laboratory.

The client must record the time of semen collection and deliver the sample to the laboratory within 1 hour.

When transporting to the laboratory, the sample must be kept between 20 °C and 37 °C.

The report should include that the sample was collected by means of a special condom during sexual intercourse at home or another location outside the laboratory.

Note: Ordinary latex condoms should not be used for semen collection because they contain agents that interfere with the motility of spermatozoa.

NB:

1. Coitus interruptus is not a recommended for semen collection,
   This is because the first portion of the ejaculate, which has the highest number of spermatozoa, can be lost. and also, there may be cellular and bacteriological contamination of the semen sample, and the low pH of the vaginal fluid could adversely affect sperm motility.
2. If the client cannot obtain a semen sample, the postcoital test may provide some information about his spermatozoa.

Safe handling of specimens

• Semen samples can harbor dangerous infectious agents (like human immunodeficiency virus (HIV), hepatitis viruses or herpes simplex virus) and must handled as a biohazard.
• Safety should be strictly adhered to e.g. good laboratory  practice is fundamental to laboratory safety.

Delayed liquefaction: Usually samples may not liquefy, making semen evaluation difficult. In these cases, additional treatment, mechanical mixing or enzymatic digestion should be done. Some samples may be induced to liquefy by adding an equal volume of physiological medium (e.g. Dulbecco’s phosphate-buffered saline;  followed by repeated pipetting.
Inhomogeneity can be reduced by repeated (6–10 times) gentle passage through a blunt gauge 18 (internal diameter 0.84 mm) or gauge 19 (internal diameter 0.69 mm) needle attached to a syringe. Digestion by bromelain, a broad-specificity proteolytic enzyme can help promote liquefaction.

(b). Appearance:

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

(c). Viscosity:

After ejaculation, normal semen is thick and viscous. It liquefies within 30 minutes by the action of proteolytic enzymes secreted by prostate. If liquefaction does not happen within 60 minutes, it is abnormal. Viscosity of the semen 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.

(d) Volume:

Volume of ejaculate is normally greater than 2 ml. It is measured after liquefaction. Volume  less than 2.0 ml is abnormal, and is associated with low sperm count. The volume is best measured by weighing the sample in the vessel in which it is collected  as follows:

• Obtain the sample in a pre-weighed, clean, disposable container.
• Take the weigh of the vessel with semen in it.
• Subtract the weight of the container.
• Compute the volume from the sample weight, assuming the density of semen to be 1 g/ml (Auger et al., 1995). (Semen density varies between 1.043 and 1.102 g/m.

NB:

• Empty specimen containers usually have different weights, so each container must be individually pre-weighed and recorded on the container before it is given to the client.
  Alternatively, the volume can be measured directly. Collect the sample directly into a modified graduated glass measuring cylinder with a wide mouth. These can be obtained commercially. Read the volume directly from the graduations (0.1 ml accuracy).
• Measuring volume by aspirating the sample from the specimen container into a pipette or syringe, or decanting it into a measuring cylinder, is not recommended, because not all the sample will be retrieved and the volume will therefore be underestimated. Sample loss can be between 0.3 and 0.9 ml
• Low semen volume is characteristic of obstruction of the ejaculatory duct or congenital bilateral absence of the vas deferens. condition in which the seminal vesicles are also poorly developed.
• Low semen volume can also be the result of collection problems (loss of a fraction of the ejaculate), partial retrograde ejaculation or androgen defi ciency.
• High semen volume may reflect active exudation in cases of active inflammation of the accessory organs.

Lower reference limit

The lower reference limit for semen volume is 1.5 ml

(e). pH:

Add a drop of liquefied semen sample on a  pH paper (of pH range 6.4-8.0) and pH recorded after 30 seconds. Normal semen pH is 7.2 to 8.0 after 1 hour of ejaculation. The portion of semen contributed by seminal vesicles is usually basic, while portion from prostate is acidic. Low pH (less than 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).
For normal samples, pH paper in the range 6.0 to 10.0 should be used.

Procedure is as follows:

• Mix the semen sample properly
• Add a drop of the sample by spreading it evenly onto the pH paper.
• Wait for the color of the impregnated zone to become uniform (in less than 30 seconds).
• Compare the colour with the calibration strip to read the pH.

NB:

Ensure that accuracy of the pH paper should be checked against known standards. For viscous samples, the pH of a small aliquot of the semen can be measured with a pH meter designed for measurement of viscous solutions

2. Microscopic Examination

Microscopic examination of the semen is the most important in semen analysis for infertility.

This provides an overview of the sample, to reveal:

• Aggregation or agglutination;
• Mucus strand formation;
• The presence of cells other than spermatozoa, e.g. epithelial cells, “round cells” (leukocytes and immature germ cells) and isolated sperm heads or tails.
  The preparation should then be observed at ×200 or ×400 total magnification (i.e. a combination of a ×20 or a ×40 objective with a ×10 ocular).

This permits:

• Assessment of sperm motility;
• Determination of the dilution required for accurate assessment of sperm number

NB:  Mix the sample well in the original container before removing an aliquot of semen for assessment, but not so vigorously that air bubbles are created. This can be achieved by aspirating the sample 10 times into a wide-bore disposable plastic pipette. Do not mix with a vortex mixer at high speed as this will damage spermatozoa.

• Mix the semen sample well .
• Take off the aliquot of semen immediately after mixing, allowing no time for the spermatozoa to settle out of suspension.
• Remix the semen sample before removing replicate aliquots.
• The volume of semen and the dimensions of the coverslip must be standardized, so that the analyses are carried out on a preparation of fixed depth of about 20 m, which allows the spermatozoa to swim freely:
• Place a standard volume of semen, e.g. 10 l, onto a clean glass slide.
• Cover with a cover glass to provide a chamber approximately 20m deep. The weight of the coverslip spreads the sample.
• Avoid the formation and trapping of air bubbles between the coverslip and the slide.
• Analyse the freshly made wet preparation as soon as the contents are no longer drifting.

Depth of wet preparations

The depth of a preparation is calculated by; dividing the volume of the sample (Vol = mm3) by the area over which it is spread (A, mm2): D = V/A. Thus, a volume of 10 ml of semen delivered onto a clean glass slide and covered with a 22 mm × 22 mm coverslip (area 484 mm2) provides a chamber of depth of 20.7 mm; a 6.5 ml sample covered with an 18 mm × 18 mm coverslip (area 324 mm2) provides a depth of 20.1 mm; an 11 ml sample covered by a 21 mm × 26 mm coverslip (area 546 mm2) provides a depth of 20.1 mm.

Occasionally, a deeper chamber may be required: a 40 ml sample covered by a 24 mm × 50 mm coverslip (area 1200 mm2) = provides a depth of 33.3 mm.

Note:

A chamber depth of below 20 mm constrains the rotational movement of spermatozoa.

If the chamber is very deep, it will be hard to assess spermatozoa as they move in and out of focus

If the number of spermatozoa per visual field varies considerably, the sample is not homogeneous. In such cases, the semen sample should be mixed again thoroughly and a new slide prepared as above.

Lack of homogeneity may also result from abnormal consistency, abnormal liquefaction, aggregation of spermatozoa or sperm agglutination

Aggregation of spermatozoa

This is the adherence either of immotile spermatozoa to each other or of motile spermatozoa to mucus strands, non-sperm cells or debris. This is considered to be nonspecific aggregation and must be recorded.

This refers to motile spermatozoa sticking to each other, head-to-head, tail-to-tail or in a mixed way. The motility is usually vigorous with a frantic shaking motion, but sometimes the spermatozoa are so agglutinated that their motion is limited. Any motile spermatozoa that stick to each other by their heads, tails or midpieces should be noted.

The major type of agglutination (grades 1–4) and the site of attachment (grades A–E) must be recorded;

• Grade 1: These are isolated less than 10 spermatozoa per agglutinate, many free spermatozoa
• Grade 2: Theses are moderate between 10–50 spermatozoa per agglutinate, free spermatozoa
• Grade 3: Theses are  large agglutinates of more than 50 spermatozoa, some spermatozoa still free
• Grade 4: Theses are gross all spermatozoa agglutinated and agglutinate interconnected

Note: Motile spermatozoa stuck to cells or debris or immotile spermatozoa stuck to each other (aggregation) should not be scored as agglutination.

Categories of sperm movement:  Grading motility is recommended as it distinguishes spermatozoa with progressive or non-progressive motility from those that are immotile. The motility of each spermatozoon is graded as follows:

• Progressive motility (PR): These move actively, either linearly or in a large circle, regardless of speed.
• Non-progressive motility (NP): These have all other patterns of motility with an absence of progression, e.g. swimming in small circles, the flagellar force hardly displacing the head, or when only a flagellar beat can be observed.
• Immotility (IM): These have no movement at all.

(b) Sperm Viability or Vitality

Principle: A cell with intact cell membrane (a vital or viable cell) will not take up the eosin Y and will not stain, while a non-viable or dead cell with damaged cell membrane, will take up the dye, and will be stained pink-red. Another stain (nigrosin) may be used to stain the background material. The test is performed if motility is abnormal.

Procedure

• Mix one drop of semen with 1 drop of eosin-nigrosin solution and incubate for 30 seconds.
• A smear is made from a drop placed on a glass slide.
• The smear is air-dried and examined under oil immersion objective. White sperms are classified as live or viable, and red sperms are classified as dead or non-viable. At least 200 spermatozoa are examined.
• 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.

(c)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).

Procedure

• Semen is diluted 1:20 with sodium bicarbonateformalin diluting fluid (Take 1 ml liquefied semen in a graduated tube and fill with diluting fluid to 20 ml
  mark. Mix well).
• A cover glass 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 spermatozoa to settle.
• The chamber is mounted on the microscope stage.
• Using the 20× or 40× objective and iris diaphragm lowered sufficiently to give sufficient contrast, number of spermatozoa is counted in 4 large corner squares. Spermatozoa whose heads are touching left and upper lines of the square should be considered as ‘belonging’ to that square.
• Sperm count per ml is calculated as follows:

(d) Sperm Morphology

Here, a smear is prepared by spreading a drop of semen 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 has of three main components: head, neck, and tail. Tail is further subdivided into midpiece, main (principle) piece, and end piece. 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 membrane bound vesicle containing glycoproteins and enzymes. These enzymes are important in separation of cells of corona radiata and dissolution of zona pellucida of ovum in the process of fertilization.

The 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.

Atleast more than 30% of spermatozoa should show normal morphology. The defects in morphology that are associated with infertility in males include:

• Defective mid-piece which causes reduced motility
• An incomplete or absent acrosome which causes inability to penetrate the ovum,
• A giant head (defective DNA condensation).

Normal sperm morphology

• Total length of sperm: About 60 μ
• Head: Length: 3-5 μ, Width: 2-3 μ, Thickness: 1.5 μ
• Neck: Length: 0.3 μ
• Middle piece: Length: 3-5 μ, Width: 1.0 μ
• Principal piece: Length: 40-50 μ, Width: 0.5 μ
• End piece: 4-6 μ

Abnormal morphology
1. Abnormalities in the head:

• Large heads
• Small heads
• Pyriform heads
• Round heads
• Amorphous heads
• Vacuolated heads (more than 20% of the head area occupied by vacuoles)
• Small acrosomes (occupying less 40% of head area)
• Double heads
• Tapered heads
• Pyriform heads
• Round heads

3. Abnormalities in the neck:

• Bent neck and tail forming an angle more 90° to the long axis of head

4. Abnormalities in the piece:

• Asymmetric insertion of midpiece into head
• Thick or irregular midpiece
• Abnormally thin midpiece

5. Abnormalities in the in tail:

• Irregular tails
• Multiple tails
• Tails with irregular width
• Bent tails
• Short tails
• Coiled tails

6. Pin heads: Not to be counted

7. Cytoplasmic droplets

• Greater than 1/3rd the size of the sperm head

8. Precursor cells: Considered abnormal
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 greater than 1 million/ml indicate:

• presence of infection.

Presence of large number of immature sperm cells:

• Indicates spermatogenesis dysfunction at the testicular level.

3. Immunologic Analysis

Antisperm Antibodies

The immunological tests done on seminal fluid are; 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.

However, the role of Antisperm antibodies in the cause of male infertility is controversial.

a). SpermMARTM test: This test detects IgGs and IgAs antibodies against sperm surface in semen sample.

1. In direct SpermMARTM IgG test: In this 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. And 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..
2. In direct SpermMARTM IgA test: In this 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.
3. In indirect SpermMARTM: In this test, 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  more than 50% of spermatozoa show attached latex particles, immunological problem is likely.

b). Immunobead test: Here, Antibodies bound to the surface of the spermatozoa may 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 more than 50% spermatozoa with attached beads is abnormal.

4. Biochemical Analysis of Semen

Biochemical markers are usually done to measured secretions of accessory structures. These include fructose (seminal vesicles), zinc, citric acid or acid phosphatase (prostate), and α-glucosidase or carnitine (epididymis).

Test for Fructose

Resorcinol procedure is employed for detection of fructose where 5 ml of resorcinol reagent 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.

Sperm Function Tests or Functional Assays

Postcoital (Sims-Huhner) Test

This involves 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. If more than 10 motile sperms are observed the test is considered as normal.

An abnormal test may result from:

• Poor quality of cervical mucus due to wrong judgment of ovulation,
• lack of ejaculation,
• poor semen quality,
• cervicitis
• treatment with antioestrogens (e.g. Clomid),
• Absence of motile sperms due to ineffective technique of coitus,
• use of coital lubricants that damage the sperm,
• 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

How to interpret postcoital 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-like manner.
• Abnormal: Absence of sperms or large number of sperms are dead or sperms are clumped; cervical mucus cannot stretch 2 inches (5 cm) or does not dry in a fern-like manner.

Cervical Mucus Penetration Test

Here, the 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 more than 30 mm, while most infertile men show scores less than 20 mm.

Hamster Egg Penetration Assay

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;

• Number of eggs penetrated (penetration rate less 15% indicates low fertility),
• Number of sperm penetrations per egg (Normal greater 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 looks for 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 involves 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 rape 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 of clothing from stained portion is soaked in 1-2ml of physiologic saline for 1 hour. A similar piece of clothing distant from the stain is also soaked in saline as a control.

Tests

• Microscopic examination for sperms: Presence of motile sperms in vaginal fluid indicates interval of less than 8 hours. Smears prepared from collected samples are stained and examined for the presence of sperms.
• 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 at least 50 U/sample is considered as positive evidence of semen.
• 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.
• 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 to high choline content of some other body fluids.

Semen analysis to check the effectiveness of vasectomy

The reason for 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.

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