SPERM EVALUATION AND TESTING

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Sperm Evaluation and Testing

Sperm Counts

Laboratories performing sperm " counts " , in general, vary in the details that

they provide the physician requesting the " count " . A general sperm count as

part of a fertility evaluation should include the total density or count (20

million per ml or above), and the motile density (8 million per ml or higher).

The motile density is perhaps the most important part of the semen analysis, as

it reports the total number of sperm thought capable of progressing from the

site of sperm deposition to the site of fertilization. This value is essential

in both allowing a determination regarding whether or not a semen analysis is

" normal " , as well as in providing prognostic information should advanced

reproductive medical assistance be required.

Definitions of " abnormal " counts:

• Polyzoospermia: Excessively high sperm concentration.

• Oligozoospermia: Sperm count less than 20 million/ml

• Hypospermia: Semen volume < 1.5 ml

• Hyperspermia: Semen volume > 5.5 ml

• Aspermia: No semen volume

• Pyospermia: Leukocytes (germ fighter cells) present in semen

• Hematospermia: Red blood cells present in semen

• Asthenozoospermia: Sperm motility < 40%

• Teratozoospermia: > 40% of sperm seen are of abnormal form

• Necrozoospermia: Nonviable ( " dead " ) sperm

• Oligoasthenozoospermia: Motile density < 8 million sperm/ml

Sperm Morphology (Shape and Appearance)

The evaluation of sperm size, shape and appearance characteristics should be

assessed by carefully observing a stained sperm sample under the microscope. The

addition of colored " dyes " (stains) to the sperm allow the observer to

distinguish important normal landmarks (characteristics) as well as abnormal

findings. Several methods of staining sperm are used, and the method employed

should be one with which the examiner is comfortable and experienced.

Several different shapes or forms of human sperm have been identified and

characterized. These forms fall into one of four main categories: normal forms,

abnormal head, abnormal tail and immature germ cells (IGC).

Normal forms

Normal sperm have oval head shapes, an intact central or " mid " section, and an

uncoiled, single tail.

Abnormal heads

Many different sperm head abnormalities may be seen. Large heads

(macrocephalic), small heads (microcephalic) and an absence of identifiable

head are all seen in evaluations. Tapering sperm heads, pyriform heads

(teardrop shape) and duplicate or double heads have been seen. Overall (gross)

abnormalities in appearance may be termed " amorphous " changes.

Abnormal tails

Coiling and bending of the tail are sometimes seen. Broken tails of less than

half normal length should be categorized abnormal. Double, triple and quadruple

tails are seen and are abnormal. Cytoplasmic droplets along the tail may

indicate an immature sperm.

Immature germ cells (IGC's)

White blood cells (WBC's, germ fighters) in the semen should rarely be seen. It

is very difficult to distinguish between an immature germ cell and a WBC.

Because the presence of WBC's in the semen (pyospermia) can be a serious

concern, if a report of " many IGC's " is delivered, it becomes very important to

assure that these cells are not, instead, WBC's.

Sperm " Motility " (Movement)

Sperm motility studies identify the number of motile (moving) sperm seen in an

ejaculate specimen. Here again, as in many other sperm studies, many

laboratories use " normal " values that are out of date and inaccurate. Many labs

will assess sperm motility upon receipt of the specimen, and again at hourly

time intervals for four to twenty four hours. It is well known that sperm

motility is a temperature dependent sperm function, so the handling and

processing of specimens is critical. It is for this reason that we, except in

very rare instances, require that specimens be evaluated only in a laboratory

such as our own, where we are able to tightly control laboratory conditions. We

have found the repeated testing of sperm over time for motility adds little to

the evaluation of motility over the initial sperm motility assessment. Sperm

are known not to survive well for extended periods of time in semen, and in

nature, sperm very rapidly leave the semen to enter the cervical mucus. Many

laboratories consider " normal " sperm motility to be 60% or greater. Our own

studies, in agreement with many others have found men with 40% or greater sperm

motility to be " normal " .

Asthenozoospermia

Decreased sperm motility. If found to be present, exam should be repeated to

assure that laboratory conditions did not cause the problem. Frequent causes:

abnormal spermatogenesis (sperm manufacture), epididymal sperm maturation

problems, transport abnormalities, varicocele. These conditions should all be

looked for if sperm motility is repeatedly " low " .

Necrozoospermia

A total absence of moving sperm. It is vital, if sperm are seen, but are not

moving, to carry out studies (vital stains) to see if the sperm seen are alive.

It is possible to have sperm with normal reproductive genetics that are

deficient in one or several of the factors necessary to produce motility. We

have achieved several successful pregnancies emploting microinjection of

healthy, non motile sperm directly into the egg (ICSI).

Chemical and Biochemical Semen Characteristics

Semen acid-base balance (pH)

The pH of semen is measured using a specially treated paper blot that changes

color according to the pH of the specimen that it is exposed to. The pH of

normal semen is slightly alkaline ranging from 7.2 to 7.8. Prostatic secretions

are acidic while the secretions of the seminal vesicles are alkaline.

Therefore, alterations in pH may reflect a dysfunction of one or both of these

accessory glands. The pH of semen has not been generally found to have a major

influence on a man's fertility potential.

Color and Turbidity

Semen is normally translucent or whitish-gray opalescent in color. Blood found

in semen (hematospermia) can color the semen pink to bright red to brownish

red. The presence of blood in semen is abnormal and should be reported. The

presence of particles, nonliquified streaks of mucus or debris requires further

evaluation.

Liquefaction

Semen is normally produced as a coagulum. The specimen will usually liquefy

within 30 minutes. The failure to liquefy within one hour is abnormal.

Excellent methods for correcting this problem in the laboratory are available.

Viscosity

Nonliquefaction and excessive viscosity are two separate conditions. Viscosity

is measured after complete liquefaction has occurred. Viscosity is considered

" normal " if the liquefied specimen can be poured from a graduated beaker drop

by drop with no attaching agglutinum between drops. The role of hyper

(excessive) viscosity is being studied, but it seems possible that this

condition may interfere with the ability of sperm to travel from the site of

deposition into the cervix or uterus.

Computer Assisted Semen Analysis (CASA)

The use of computer assisted semen analysis has advanced the ability to study

and understand sperm function as it relates to human infertility. The major

advances have been in the ability to more accurately determine sperm

concentration (counts) and motility (movement). Generally, sperm are " looked "

at by a computerized digitizing tablet through a microscope. The computer has

been " taught " by the laboratory personnel what sperm look like, and how they

move. When the computer then " sees " a sperm under the microscope, it is able to

draw a digitized picture of each individual sperm, including the speed and path

this sperm takes while moving under the microscope. A great deal has been

learned about the normal and abnormal " micro " characteristics of sperm employing

this method. The method is, however, not foolproof. The computer is only as

intelligent as it's programmer. Small changes in the computer program can alter

the sperm calculations significantly. The computers must constantly be

monitored and updated. In our laboratories, all grossly abnormal CASA assays

are always verified by both a repeat analysis as well as with a " hands on "

human second look opinion. We feel that any abnormal sperm count must be

verified by a manual counting and assessment method.

Sperm Penetration Assays (SPA, " Hamster Tests " )

There have been many attempts made to develop a Laboratory test that will

accurately predict the ability of a human sperm to fertilize a human egg. Dr.

Aitken and his group many years ago demonstrated a correlation between sperm

movement characteristics and sperm fertilizing ability as evaluated by the zona

pellucida-free hamster egg penetration test. In this test, the species specific

barrier to penetration (not fertilization) is removed from the ova (eggs) of

the hamster. These oocytes are then exposed to prepared sperm from the man

being tested. There is some feeling that if a man's sperm are able to penetrate

the hamster eggs in the laboratory, there is a higher likelihood that his sperm

will ultimately be able to fertilize a human egg if so exposed. This test is

not uniformly accepted, due to the high false negative (no penetration of the

hamster egg, but wife gets pregnant anyway) rate and the sometimes seen false

positive (penetrates the hamster egg but does not fertilize human eggs in

vitro) rate of this test. Our experience has been that good performance in the

hamster test can provide some limited reassurance of the likelihood that a

man's sperm will be able to achieve fertilization if given the chance. If men

fail the hamster test, we rely upon in vitro fertilization with ICSI. This

protocol has provided us with excellent success rates in men whose sperm

function remains questionable. It should be noted that most men that fail the

hamster test, are able to achieve normal fertilization with ICSI.

Post-Coital Testing

The postcoital test (also known as the Huhner test or the Sims-Huhner test) is

a valuable office test that should be carried out in selected patients early in

their infertility evaluation. While this is a very popular and widely used

test, there are no widely accepted normal values for the interpretation of this

test. Simply, the postcoital (after intercourse) test evaluates the women's

cervical mucus at the time of ovulation and how the mucus interacts with her

husband's sperm as ovulation is about to occur. The couple is instructed to

avoid sexual intercourse for two days prior to the exam. When evidence of

impending ovulation is detected (LH testing, hormone blood tests, ultrasound,

etc.) the couple is instructed to have intercourse and then present to the

office 6 to 10 hours later (standard test). At this time, a small drop of mucus

is painlessly removed from the endo (inner) cervix, and this drop is examined

under the microscope. A favorable result would find many sperm in thin watery

mucus, with good forward, active motion through the mucus. If the initial test

is good, a second delayed exam (18-24 hours after intercourse) may be required

if infertility persists. If the initial test is poor, a repeat exam carried out

2-3 hours after intercourse may be needed. The timing of the postcoital exam is

very important. If carried out too soon after intercourse, sperm that appear

normal at that time may later die, giving a false sense of security. Patients

should assure that the test timing is appropriate, and that they are not just

being squeezed in to a busy schedule at a convenient time. A normal test

largely excludes the cervix as a contributor to any fertility problem.

Sperm Washing and Freezing

Sperm " washing " techniques have been applied to treat a wide variety of sperm

and semen disorders, as well as to prepare " normal " sperm for intrauterine

insemination in the treatment of some female disorders. What is being " washed "

in a sperm washing procedure are the various constituents of semen and the

remainder of the ejaculate not deemed necessary to achieve fertilization of the

egg. An ejaculate is not a sterile specimen and may contain both aerobic

(oxygen dependent) and anaerobic bacteria. In addition cellular debris from

the vas deferens, the prostate, the seminal vesicles and the urethra may be

present. All of these components are " washed " from the specimen in the sperm

wash procedure.

Antisperm Antibodies

Antisperm antibodies have been well documented in the scientific literature as

having the potential to cause impairment of fertility in humans. Sperm

antibodies are detectable in either the male or female partner in approximately

10% of infertile couples. While these antibodies may be present, they may not

be ultimately implicated as the cause of the infertility, making the search for

antibodies in infertile couples both important and frustrating for the

physician.

Antibodies, in general, are biochemical " time-bombs " that develop in the immune

systems of all normal human beings. They are there to protect us from foreign

" invaders " (viruses, bacteria, foreign objects, etc.) that would otherwise have

the potential to attack and harm vital parts of the body. A newborn infant is

supplied with a temporary supply of vital antibodies from the mother. This

supply may be initially be replenished and transmitted from breast milk from

the mother. Ultimately, antibodies to harmful outsiders develop slowly and

reliably over time as a growing human is exposed to more and more " coughs,

clods and flu's " from the outside world. Vaccines are a way to trick the body

into producing long term protective antibodies without the body having to first

suffer the disease. Antibody protection can, on occasion, " short circuit " . In

these instances, the abnormal function of the antibodies can lead to a variety

of diseases. Some common examples are certain forms of severe arthritis, lupus,

diabetes, and in reproduction, premature menopause (ovarian failure) and

antisperm antibodies.

What happens in many immunologic disorders is the immune system that is

normally ONLY supposed to make antibodies to protect from harmful threats

begins to see " normal " tissue as a threat. In the case of arthritis, the immune

system mistakenly decides that a person's bone joints have become a threat and

begins to attack the joints. This persistent immune attack leads to an eventual

painful destruction of the involved joints. In the case of antisperm

antibodies, either the man begins to see his own sperm as a foreign " threat " or

his female partner, whose immune system is supposed to tolerate sperm as

non-threatening, begins to lose this tolerance and produces a destructive

antibody that may damage the sperm and make it incapable of performing it's egg

penetration and fertilization duties.

Antisperm antibody testing is complex, as at least three different antibodies

can have a damaging effect on sperm. Each of these antibodies must be

specifically looked for in the investigation of the male and female. A new

test, due for release to laboratories in early 2001 is able to determine

biochemically if sperm have been damaged by any cause within the male

reproductive tract, making them incapable of fertilizing the egg. This new

assay promises to make our ability to assess sperm function much more accurate.

Initial use of this investigation assay has shown it to be nearly 100% accurate

in determining is a sperm can fertilize an egg without help, either naturally

or with in vitro fertilization, of if intervention in the fertility laboratory

with ICSI will be required.

Hart

Wife to Jon, Love of My Life

Mom to 4:

Arianne (16) ~ a(7)

(4) ~ (My TR Baby - born 6/20/02)

Glory to God and Many Thanks to Dr. Levin

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