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Semen test-Sperm analyzer

Semen test-Sperm analyzer

MICROSCOPIC EXAMINATION

Sperm concentration

A phase contrast microscope using volumetric dilution and hemocytometry is recommended for all examinations of unstained preparations of fresh/washed semen and is reported as millions of sperm per mL. Samples in which no sperm are identified should be centrifuged and the pellet examined for the presence of sperm. Pregnancy rates by intercourse and intrauterine insemination decline as sperm density decreases.

Azoospermia refers to the absence of sperm in the seminal plasma.

Oligozoospermia (also often called oligospermia) refers to seminal plasma concentration less than 20 million per milliliter.

Motility

The efficient passage of spermatozoa through cervical mucus is dependent on rapid progressive motility,that is, spermatozoa with a forward progression of at least 25 μm/s. Reduced sperm motility can be a symptom of disorders related to male accessory sex gland secretion and the sequential emptying of these glands.

Rapid and slow progressive motility is calculated by the speed at which sperm moves with flagellar movement in a given volume as a percentage (range 0%-100%) by counting 200 sperms.

a. 

Rapid progressive motility (ie, >25 μm/s at 37°C and >20 μm/s at 20°C; Note: 25 μm is approximately equal to 5 head lengths or half a tail length).

b. 

c. 

Slow or sluggish progressive motility

d. 

e. 

Nonprogressive motility (<5 μm/s)

f. 

g. 

Immotility

h. 

A normal semen analysis must contain at least 50% grade A and B, progressively motile spermatozoa. If greater than 50% sperms are immotile then the sperms should be checked for viability. Persistent poor motility is a good predictor of failure in fertilization, an outcome that is actually more important when making decisions regarding a couple's treatment options.

Morphology

The clinical implications of poor morphology scores remain highly controversial. The initial studies using rigid criteria reported that patients undergoing in vitro fertilization (IVF) who had greater than 14% normal forms had better fertilization rates. Later studies reported that most impairment in fertilization rates occurred with morphology scores of less than 4%.

The staining of a seminal smear (Papanicolaou Giemsa, Shorr, and Diff-Quik) allows the quantitative evaluation of normal and abnormal sperm morphological forms in an ejaculate. Smears can be scored for morphology using the World Health Organization (WHO) classification, or by Kruger's strict criteria classification. WHO method classifies abnormally shaped sperm into specific categories based on specific head, tail, and midpiece abnormalities, which is based on the appearance of sperm recovered from postcoital cervical mucus or from the surface of zona pellucida (>30% normal forms). In contrast, Kruger's strict criteria classifies sperm as normal only if the sperm shape falls within strictly defined parameters of shape and all borderline forms are considered abnormal (>14% normal forms).

a. 

Head defects: Large, small, tapered, pyriform, round, amorphous, vacuolated (>20% of the head area occupied by unstained vacuolar areas) heads with small acrosomal area (<40% of head area), double heads, any combination of these.

b. 

c. 

Neck and midpiece defects: Bent neck; asymmetrical insertion of midpiece into head; thick, irregular midpiece; abnormally thin midpiece; any combination of these.

d. 

e. 

Tail defects: Short, multiple, hairpin, broken, bent, kinked, coiled tails, or any combination of these.

f. 

g. 

Cytoplasmic droplets: Greater than one-third of the area of a normal sperm head.

h. 

Morphology should be used along with other parameters, and not as an isolated parameter, when determining clinical implications. It is important to realize that, in general, pregnancy is possible with low morphology scores and that both motility and morphology have also demonstrated prognostic value, as do combinations of parameters.

LEUKOCYTOSPERMIA—FIGURE

Infection of the male reproductive tract can directly or indirectly cause infertility.Pyospermia is a laboratory finding categorized as the abnormal presence of leukocytes in human ejaculate and may indicate genital tract inflammation.

To differentiate round cells from polymorphonuclear (PMN) leukocytes, which are primary sources of reactive oxygen species (ROS) generation, peroxidase staining is used. Neutrophils, polynuclear leukocytes, macrophages are peroxidase-positive granulocytes (PMN should be 1 × 10 6 /mL), whereas degranulated PMNs, lymphocytes, and “immature” germ cells are peroxidase negative.

SPERM AGGLUTINATION—ANTISPERM ANTIBODY

Immunologic protection to sperm antigens are provided by the tight junctions of sertoli cells forming the blood-testis barrier. The spermatozoon evokes an immune response when exposed to the systemic immune defense system in conditions in which this barrier gets disrupted, leading to the formation of antisperm antibodies (ASA). Certain ASAs have a cytotoxic effect on the spermatozoa and can cause cell death and immobilization of sperm cells. Other effects of ASAs include creating agglutinated clumps of moving sperm in the semen sample, hampering passage of sperm through the cervical mucus, and zonal binding and passage.

Two current methods of detecting antibodies bound to the surface of motile sperm are the mixed agglutination reaction assay (MAR test; only for IgGs) and the immunobead-binding assay (for IgA, IgG, and IgMs).A positive finding of >50% of motile sperm with attached beads is considered to be clinically significant, but with the advent of assisted reproduction technology (ART), ASA testing has lost its relevance.

BIOCHEMICAL ASSESSMENT OF SEMINAL PLASMA, PROSTATE, EPIDIDYMIS, AND SEMINAL VESICLES

Biochemical assessment is carried out to assess the impairment of epididymal, vesicular, and prostatic function and it may be clinically relevant in patients with hyperviscous semen and to understand genital fluid interactions during the semen coagulation-liquefaction process. Decreased levels of zinc, citric acid, and glucosidase may indicate either seminal vesicle, prostate dysfunction, or prostatic duct obstruction.

SPERM FUNCTIONAL TESTS

Clinicians are still searching for semen parameter thresholds in the so-called normal fertile populations to be able to define fertility, subfertility, and infertility more accurately. Notwithstanding such lack of uniform criteria, if sperm abnormalities are observed in the “basic” semen analysis or if the couple is diagnosed as “unexplained” infertility, the workup should proceed to the analysis of sperm functional tests (second-tier level). The diagnosis of subfertility or infertility, based on the first-tier (initial “basic” evaluation) and the “expanded” screening or second-tier level (functional), will direct management toward a variety of therapeutic options.To accurately use the functional assays, the clinician must understand what the test measures, what the indications are for the assay, and how to interpret the results to direct further testing or patient management. It is at this time that sperm function/biochemical tests may be of highest value to direct the couple to ART. Assisted reproduction can be indicated as a result of (1) failure of urologic/medical treatment, (2) the diagnosis of “unexplained” infertility in the couple, (3) the presence of “basic” sperm abnormalities of moderate-high degree, or (4) abnormalities of sperm function as diagnosed by predictive bioassays of the “expanded” screening.

SPERM CERVICAL MUCUS INTERACTION

The postcoital test (PCT) evaluates the sperm-cervical mucus interaction and the presence of more than 10-20 sperm per 400 high-power fields, the majority of which demonstrate progressive motility, is usually considered normal. The finding of immobilized sperm with a side-to-side shaking motion suggests the presence of antisperm antibodies either on the sperm or in the cervical mucus. An abnormal PCT result suggests, but does not prove, cervical factor infertility. As timing of PCT is problematic, in vitro penetration tests, probably using mucus substitutes, such as methyl cellulose or hyaluronic acid, provide an alternative that has shown promise but require more extensive validation.

1. 

Computer-assisted semen analysis

2. 

3. 

Sperm viability testing

4. 

5. 

Tests of sperm capacitation

6. 

7. 

Tests of hemizona and zona pellucida binding

8. 

9. 

Sperm penetration assay or sperm capacitation index or zona-free hamster oocyte penetration assay

10. 

11. 

Tests of sperm DNA damage

12. 

13. 

Assessment of ROS

14. 

15. 

Sperm proteomics

16. 

COMPUTER ASSISTED SEMEN ANALYSIS

Manual semen analysis lacks the ability to measure the kinematics of sperm motion. CASA is potentially useful because of its capacity to analyze sperm motion (sperm head and flagellar kinetics), some of which have been shown to be related to IVF outcome.

Some of the important kinematic parameters are as follows:

i. 

Curvilinear velocity: Curvilinear velocity (VCL) is the measure of the rate of travel of the centroid of the sperm head over a given time period.

ii. 

iii. 

Average path velocity: Average path velocity(VAP) is the velocity along the average path of the spermatozoon.

iv. 

v. 

Straight-line velocity: Straight-line velocity (VSL) is the linear or progressive velocity of the cell.

vi. 

vii. 

Linearity: Linearity of forward progression (LIN) is the ratio of VSL to VCL and is expressed as percentage.

viii. 

ix. 

Amplitude of lateral head displacement: Amplitude of lateral head displacement (ALH) of the sperm head is calculated from the amplitude of its lateral deviation about the cell's axis of progression or average path.

x. 

Motility

Although CASA is very accurate for determining the details of sperm kinetics, manual assessment of semen is much more accurate in discerning among debris, crystals, and immotile, dead sperm heads. Therefore, manually assessed sperm concentrations and number of immotile spermatozoa are much more reliable than corresponding data obtained by CASA, provided individual is adequately trained with appropriate internal and external quality control measures.

Viability assays

Sperm viability testing is used to determine if nonmotile sperm are alive or dead and are indicated when sperm motility is less than 5%-10%. They are useful in primary ciliary dyskinesia where ultrastructural defects in sperm flagella result in absent or extremely low motility but with high viability. Also used to select sperm for intracytoplasmic sperm injection (ICSI), in surgically retrieved testicular tissue, sperms are alive but generally nonmotile, because of lack of epididymal transit. Viability testing is done by dye exclusion assays or hypoosmotic sperm swelling (HOS test).

Dye exclusion assays rely on the ability of live sperm to resist absorption of certain dyes, whereas these dyes penetrate and stain nonviable sperm. Trypan blue and Eosin Y stains, which do not stain live sperm, are commonly employed. However, as the technique requires air drying after staining, sperms are killed and not practically useful.

In HOS test when live cells are placed in hypoosmotic media, water enters the cytoplasm causing the cell to swell, particularly the tail, which is calculated as a percentage. This assay does not damage or kill the sperm and is very useful for identifying viable, nonmotile sperm for ICSI. HOS has a limited ability to predict male fertility, but an HOS result <50% is associated with increased miscarriage rates.


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