Sperm Morphology And Extended Testing

Question 1

How is the definition of normal morphology arrived at?

Answer 1

Diagnostic semen analysis is the assessment of an extremely heterogenous cohort of sperm. The definition of normal is derived from examining what samples lead to pregnancy and determining what the sperm that lead to conception look like. Other studies have looked at the morphology of the sperm found at the site of fertilisation and bound to the egg and defined some normal criteria.

Definitions must be based on strong clinical research data.

Question 2

Who found that fertility rate is significantly reduced if less than 10% of the sperm in the sample are of normal morphology?

Answer 2

Liu and Baker in 1992; IVF

Question 3

What did the study conducted by Guzick et al (2001; Natural Conception) conclude?

Answer 3

Guzick et al studied 765 infertile and 696 fertile couples. It was a large follow up study. It showed that couples with more than 12% of normal sperm in their sample had a normal chance of pregnancy, 9-12% had an intermediate chance and less than 9% had a lower chance of pregnancy.

Question 4

A study carried published in 2000 followed 210 couples over 12 menstrual cycles to fecundity. It showed a decline in natural fertility when abnormal sperm were more than 90%. Who conducted this study?

Answer 4

Zinamen et al.

Question 5

What level of sperm morphology is currently considered normal?

Answer 5

WHO (2010) suggested that the reference value below which spree morphology is definitely abnormal is at 4% (I.e. You are considered normal if you have 4% or more normal forms in your sample).

Question 6

How is sperm morphology defined?

Answer 6

Researchers have looked at sperm found in post coital cervical mucus, sperm found on the ampullary region of the Fallopian tube and sperm bound to the zona pellucida. They have shown that these sperm are much more homogenous and regular in shape than sperm found in a semen sample. Researchers have then gone on to define normal morphology.

1) . Head - single, oval, symmetrical. Approximately 5x3um. Very few vacuoles.
2) . Midpiece - slim, regular. Very few (if any) cytoplasmic droplets.
3) . Tail - single, centrally inserted. Length is 8-10x the head length at approximately 45um long. The achrosome makes up 40-70% of the head.

Certain processing techniques may affect sperm dimensions.

Clearly abnormal forms are easy to identify. Identification for normal can be more difficult. Criteria should be applied rigorously. All borderline forms should be considered abnormal, but there is variation of normal. Obvious mid-piece and tail defects are easy to spot.

Question 7

How are slides prepared for assessment of sperm morphology?

Answer 7

Prepare duplicate smears using feathering or two-slide technique or use a plastic spreader&raquo_space;> allow to air dry&raquo_space;> fix immediately when dry using a fixative appropriate to the staining method.

Two staining methods are currently recommended. These are the Papanicolaou and DiffQuik methods (a modified Giemsa stain).

Question 8

How would one go about observing the slide that has been prepared for the assessment of morphology?

Answer 8

Use bright field optics (x1000 oil immersion) in fixed stained spermatozoa.

Count at least 200 sperm (x2) as either normal or abnormal. Count only whole sperm.

Report as a percentage of normal forms.

If there is a predominance of one morphological feature/defect this should be noted and reported (including pinheads and free heads). Watch out for specific sterilising defects.

There are still labs that use a wet prep method where sperm are immobilised and examined using very high power phase contrast optics. This method first needs to be validated against a stained method if it is to be used.

An eyepiece graticule (grid) should be used to estimate head size. You don’t need to measure every sperm to the nearest nm, it is simply there to be an estimate.

If all else fails with morphology analysis then specific serialising defects should be picked up at the very least (e.g. Globoospermia, stump tail defects).

Question 9

Describe two common sterilising morphological defects.

Answer 9

1) . Globoospermia - ‘round head defect’, usually the entire population appear identical. They have no achrosomal cap on the head of the sperm.
2) . Stump tail defect - vary in length. Usually affects the entire population.

Question 10

What types of non-sperm cells may be found in the semen?

Answer 10

Round cells such as leukocytes, immature germ cells, epithelial cells and erythrocytes.

Bacteria or protazoa.

Question 11

What is the significance of seeing leukocytes in a semen sample?

Answer 11

The clinical significance of leukocytes has been studied at length but it is still unclear as to whether they have a negative or positive role in semen.

A positive role has been ascribed to white blood cells in semen and researchers have proposed that white blood cells may be there to phagocytose normal sperm or monitor infection.

More work has focussed on a potential negative role for the presence of leukocytes in semen and researchers have shown that white blood cells are responsible for the generation of free radicals (reactive oxygen species) which can impair sperm function. Infection link to sperm is unclear.

Question 12

What is the significance of seeing immature germ cells in a semen sample?

Answer 12

Immature germ cells are cells at various stages of spermatogenesis. Spermatogonia have a nuclear diameter of 6-7um, primary spermatocytes have a nuclear diameter of 8-9um, spermatids have a nuclear diameter of 4-5um.

Presence of immature germ cells is very common. Thought to be present in large numbers due to abnormal function of serotoli cells.

Clinically, research has shown an association between large numbers of serotoli cells and reduced motility and morphology of sperm and poor fertilisation at IVF.

Often associated with clinical conditions such as varicocele and hypo spermatogenesis.

Question 13

What is the significance of seeing epithelial cells on a semen sample?

Answer 13

Epithelial cells are commonly see. They are usually of urethral origin but can also be from the prostate. The clinical significance of seeing these cells in a sample is thought to be minimal.

Question 14

What is the clinical significance of seeing erythrocytes in a semen sample?

Answer 14

Erythrocytes are not normally supposed to be present in a semen sample. Their presence may be due to a surface wound around the genital area or haematospermia due to a burst blood vessel.

Question 15

How are non-sperm cells identified in a semen sample?

Answer 15

Identification of non-sperm cells can sometimes be tricky. It can be difficult to accurately distinguish germ cells on the basis of cellular morphology. However, a number of staining methods are available.

Staining methods include cyto chemical methods, immunocytochemistry methods and the Bryan-Leishman stain for germ cells.

Often non sperms cells are simply reported as non sperm cells or round cells.

Question 16

How many non sperm cells may be found on a normal ejaculate?

Answer 16

In general normal ejaculate should not contain more than 5x10^6 round cells.

Leukocytospermia is a WHO defined condition where the leukocyte concentration exceeds 1x10^6 cells/ml.

You can estimate concentration in wet prep using a haemocytometer.

Question 17

Why do we measure anti-sperm antibodies?

Answer 17

We measure anti-sperm antibodies because the formation of antibodies can be associated with reduced fertility when present in either the male or female partner.

Question 18

Why do anti-sperm antibodies occur? What is usually required for anti-sperm antibodies to develop?

Answer 18

Anti-sperm antibodies occur because sperm are a puberty event and the immune system isn’t. Immunological tolerance to self is developed in the neonate. Sperm is therefore not recognised as self and is recognised as a foreign body.

For sperm not to be recognised as self and autoimmunity to sperm to occur a breach of the blood-testis barrier is usually required. The blood testis barrier is formed by a series of tight junctions between the serotoli cells lining the tests. It is the exposure of the sperm to the outside of the testicular compartment that can lead to anti-sperm antibody immunity. It may also arise as a result of the immune cells entering the luminal compartment.

Question 19

What can cause damage to the blood-testis barrier leading to the formation of anti-sperm antibodies?

Answer 19

Damage to the blood-testis barrier can result for a number reasons. Surgery is an obvious cause. The appearance of anti-sperm antibodies after vasectomy is dependent on the technique used (35-80% variation). Patients who have vasectomy reversal (vasovasectomy) have poor sperm counts, poor motility and especially high levels of IgA. There can be very high levels of seminal antibodies in the plasma or bound to the sperm themselves.

Trauma or injury may also be a cause of damage to the blood-testis barrier.

Infection can cause damage to the barrier. For example, mumps/orchitis, varicella, chlamydia, gonococcus.

Testicular cancer is also known to be associated with the formation of anti-sperm antibodies. Foster et al (1991) found that 21% of patients with low grade tumours will form antisperm antibodies. In patients with high grade tumours 50% were found to form antisperm antibodies. It is thought that the level of antisperm antibodies is associated with disruption and disorganisation of the seminiferous tubules and the testis.

Other associations with other testicular pathologies p have a,so been made including testicular torsion, varicocele, cryptorchidism (undescended testicles) and infant inguinal hernias.

It is proposed that there is a genetic link between some of these pathologies.

Question 20

What effects do anti-sperm antibodies have on sperm?

Answer 20

Anti-sperm antibodies can impair sperm movement. Consequences include impaired sperm movement, agglutination, sperm death, inhibition of oocyte fusion and zona pellucida binding and increased rate of phagocytosis by white blood cells.

Question 21

How can we measure anti-sperm antibodies?

Answer 21

We can measure anti-sperm antibodies in blood/semen/sperm surface. 5-8% of men attending climic are thought to have IgA or IgG anti-sperm antibody in their semen. We test this using the IBT (polyacrylamide spheres) or the MAR test (uses latex particles). Both are coated with rabbit anti-IgA/IgG. These adhere to sperm coated with anti-sperm antibodies. We can then simply count the percentage of sperm bound to these particles.

Question 22

Who should be tested for anti-sperm antibodies?

Answer 22

The WHO used to recommend that everyone was tested for antisperm antibodies. These days however we need to be careful how we target resources, particularly when we know that only 5% of men will be positive and fewer still will have antibodies related to a clinically significant outcome.

Other groups have helped labs make these decisions and target resources accordingly. The royal college recommends testing samples with agglutination and/or sluggish motility. Decisions should be made with clinical colleagues. In many cases now labs will only test patients listed for IVF treatment as if IVF fails this groups are the ones most likely to lose out from not being tested in the first place.

Question 23

Why is sperm morphology important?

Answer 23

Sperm with normal morphology are better able to swim, penetrate the cervical mucus, bind to the egg and fertilise the egg.