Semen analysis

Introduction Definition of semen
– Semen is a collection of fluids which is derived from 1. Prostate gland(30%) 2. Seminal vesicles(60%) 3. Sperm canal and other secretary glands (10%) And sperm cells(spermatozoa)

The seminal vesicles:  viscid(mucoid), neutral to slightly alkaline fluid.  This fluid may be yellow or deeply pigmented depending on its flavin content  Contain substrates involved in the coagulation of semen after ejaculation.  Fructose, major nutrient for spermatozoa, is secreted  Prostate gland secretions  Is slightly acidic, milky fluid that is rich in citric acid  Also rich in acid phosphatase and in proteolytic enzymes responsible for the coagulation and liquefaction of semen

Other compounds found in the semen is ascorbic acid, prostaglandins, cholesterol, lactic acid, fibrinolytic enzymes, hyaluronidase and sperm.

Thus, function of fluids
is to have the nourishment ready to provide the energy that the sperm need. Maintain proper osmolarity and volume to neutralize the acids at the entrance to the mother's uterus, making an environment in which the sperm can move more easily.

Why we do semen analysis??? • Semen analysis is done for the following reasons:
– Evaluation of reproductive dysfunction (infertility ) in males. – Used to select donors for therapeutic insemination – To determine the effectiveness of vasectomy – In forensic studies involving alleged or suspected rape.

Collection and transport of semen Collection of semen sample after a 3-day period of continence is usually the preferred procedure.
Collection procedure 1.Explain to the patient the purpose of the test and the need for a semen specimen. 2.Instruct the patient to abstain from sexual intercourse and intake of alcohol for 2 to 3 days prior to the test.

3.Provide a clean dry, leak proof specimen container  The container should be warmed to body temperature before collection and keep this temperature until liquefaction of the coagulum is complete. •The best specimen is one collected in the primary care provider’s office or laboratory by masturbation • If the patient prefers to collect the specimen at home a silastic condom with no lubricant may be used during coitus interruptus.

N.B. A. If other plastic is used, the specimen should be examined immediately on liquefaction or transfer in to a glass container because storage in a plastic container reduces spermatozoa motility. B. The entire ejaculation must be collected for analysis because the first portion may contain most of the spermatozoa. 4. label the container (name and time of collection, period of abstinence) 5.Deliver the specimen to the lab with in 1 hour

The specimen should not be subjected to temperature extremes during transport.
– kept as near as possible body temperature.

• This is best achieved by placing the container inside a plastic bag and transporting it in the person’s armpit.

Semen analysis
Macroscopic • Physical(volume, viscosity, liquefaction) • Chemicals (PH) Microscopic • Stained preparation(determination of the percentage of normal sperm) • Wet mount(sperm count and sperm motility)

Semen analysis – When investigating infertility ,the basic analysis of semen (seminal fluid) usually includes: • Measurement of PH • Measurement of volume • Examination of wet preparation to estimate the percentage of motile spermatozoa and viable forms and to look for cells and bacteria. • Sperm count • Examination of stained preparations to estimate the percentage of spermatozoa with normal morphology

Examination of semen

• Caution=handle specimens with care b/c it may contain infectious pathogens. e.g. HIV, hepatitis virus, herpes virus Macroscopic examination Measure the volume
• Normal semen is thick and viscous with ejaculated. • It becomes liquefied usually with in 60 minutes due to fibrinolysin in the fluid • Failure to liquefy may indicate inadequate prostate secretion.

– When liquefied, measure the volume of fluid in mm using a small graduated cylinder.

• Normal specimen:usually2 to 5 mL.
– Smaller volume indicates fewer sperm and larger volume may indicate diluted fluid, again with fewer sperm than normal. – Either situation would affect fertility

• Viscosity may be observed while measuring its volume
• If viscosity is normal a semen can be poured drop by drop. • Increased viscosity may be significant if it decreases sperm motility.

Measure the PH • Using a narrow range PH paper.e. g PH 6.4 to 8.
– spread a drop of liquefied semen on the paper – After 30 seconds, record the PH – PH of normal semen: should be PH 7.2 to 7.8 – When the PH is over, this may be due to infection. – When the PH is below and the semen is found to contain no sperm, this may indicate dysgenesis ( failure to develop) of the vas deferens, seminal vesicles, or epididymis.

Microscopic examination
– Be performed to obtain estimates of sperm concentration, motility and agglutination. – Polygonal cells of the urethral tract and round cells such as spermatogenic cells and leukocytes can also be observed when sperm are counted in a hemacytometer.

• Sperm motility is a measure of the percentage of moving sperm in a sample.
– Immobile or slowly moving sperm are problematic in terms of fertility.

Estimate the percentage of motile and viable spermatozoa. Motility Procedures
– Place one drop of well mixed liquefied semen on the slide and cover with cover glass. – Focus the specimen using the 10x objective – Ensure the spermatozoa are evenly distributed. – If not, re-mix the specimen and examine a new preparation – Using the 40x objective, examine several fields.

• Evaluating the results assigns sperm to one of three catagories: – Progressive motility – Nonprogressive motility and – nonmotility • Count a normal motility: over 60% of spermatozoa are motile with in 60 minutes of ejaculation. Reporting of results – Motility (normal range 60% or above) is expressed as the percentage of sperm that move.

Grading of motility – grade 4: full activity with tail movements difficult to visualize – grade 3: good activity with tail movement visualized – Grade 2: poor to fair activity – Grade 1 : minimal forward progression – Zero progression denotes absence of any motility.

• If motility is less than 60%, a viability stain of eosin Y with negrosin as a counter stain is done. • Dead sperm will stain red, where as live sperm will exclude the dye and appear unstained. • In samples with no visible sperm, such as post vasectomy semen, the entire sample should be centrifuged and the pellet examined for intact or damaged sperm fragments.

Perform gram stain smear
– When more than 60% of spermatozoa are non motile – When more than a few leucocyte and – >6 RBC/HPF – Look for the type of bacteria that exist in the semen.

Viability • Procedure
– Mix one drop of semen with one drop of 0.5% eosin solution on a slide. – After two minutes examine microscopically. – Use the 40x objective to count the percentage of viable and non viable spermatozoa. – Viable spermatozoa remain unstained. – Non viable spermatozoa stain red – Normal viability: 75% or more of spermatozoa should be viable.

Sperm count
– Using a graduated tube or small cylinder, dilute the semen 1 in 20 as follows: • Fill the tube or thoma pipette to the 1ml mark with well mixed liquefied semen. • Add sodium bicrbonate-formalin diluting fluid to the 20 ml mark, and mix well. • Using a Pasteur pipette or thoma pipette, fill an improved Neubauer ruled chamber. • Wait 3-5 minutes for the spermatozoa to settle. • Count the number of spermatozoa in an area of 2sq mm (i.e any 2 large squares with in the 9 squares)

• Calculate the number of spermatozoa in a 1ml of fluid by multiplying the number counted by 100,000(i.e factor).
– Normal count: 20-106 x 106 spermatozoa/ml or more – Counts less than this are associated with male sterility.

• Total of 100 spermatozoa and note out of the hundred how many are motile.
– Record the percentage that are motile and nonmotile.

Analysis of sperm for size, shape, and appearance. – The more abnormal sperm present, the lower the likelihood of fertility. Staining – Make a thin smear of the liquefied well-mixed semen on a slide – While still wet, fix the smear with 95%v/v ethanol for 10 minutes. – Allow to air dry. – Wash the smear with sodium bicarbonate formalin solution to remove any mucus which may be present. – Rinse the smear with several changes of water.

– Cover the smear with dilute (1 to 20) carbolfuchsion and allow to stain for 3 minutes. – Wash off the stain with water. – Counter stain by covering the smear with dilute (1 to 20)Loeffler’s methyelene blue for 2 minutes – Wash off the stain with water – Allow the smear to air dry – Examine under 100x objective

Staining results – Nucleus of head---------------------dark blue – Cytoplasm of head-----------------pale blue – Middle piece and tail--------------pink red – Alternative stains: other staining techniques used to stain spermatozoa include giemsa and papanicolaou

Reporting morphology of spermatozoa
– Examine the preparation for normal and abnormal spermatozoa using 40 objective. – Use 100 objective to confirm abnormalities – Count 100 spermatozoa and estimate the percentage showing normal morphology and the percentage that appear abnormal.

Normal spermatozoa
– Measure 50-70µm in length – Each consist of an oval-shaped head( with acrosomal cap) which measures 3-5 x 2-3µm – A short middle piece – A long thin tail( at least 45µm in length – In normal semen, at least 50% of spermatozoa should show normal morphology. – Most specimens contain no more than20% abnormal forms.

Abnormal spermatozoa – The following abnormalities may be seen: Head • Greatly increased or decreased in size • Abnormal shape and tapering head (pyriform) • Acrosomal cap absent or abnormally large • Nucleus contains vacuoles or chromatin is unevenly distributed. • Two heads –Additional residual body, i.e cytoplasmic droplet

Middle piece
– Absent or markedly increased in size – Appears divided (bifurcated) – Angled when it meets tail

Tail
– Absent or markedly reduced in length – Double tail – Bent or coiled tail

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