Laboratory Tests

1. Routine Laboratory Tests
1.1 Semen Analysis
At first physical examination is conducted and then laboratory testing should be held. For the evaluation of the semen laboratory analysis should be held. The semen analysis does not consider being a test for fertility. Fertility determination should be held for a couple and the initiation of a pregnancy is recommended for the purpose. The best results can be achieved uniting a female factor evaluation with of the male one.
2-3 days are required to take all specimens and to evaluate them of sperm motility and forward progression within 2 hours of collection that is held at the laboratory. Specimen container should be clean, sterility is not required. Some plastics have residual spermatocidal chemicals in their content and may include spermatotoxic contaminating material as one of their constituences. The collection of the semen is held by masturbation, coitus interruptus, or with a special condom avoid of spermatocidal constituences.

“Normal” (average) and “adequate” (potentially fertile) semen quality differ. A mean or average sperm density is important because of semen quality. The results can be achieved without any difficulty. Sexual abstinence is not controlled and does not demand complete ejaculation. The age of such men is more comparing with men who have fertility evaluation.
Men with high-quality spermatozoa have fertility with very low sperm densities.

1.2. Basic Laboratory Tests
One should begin laboratory investigation of testicular function with basic screening tests. The clinical history and physical examination are the factors influencing the tests’ results. The hormones that are recommended to be tested are testosterone (T), luteinizing hormone (LH), and follicle-stimulating hormone (FSH).
Serum T is responsible for Leydig cell function and indicates intratesticular testosterone. The levels of measured circulating LH and FSH are necessary to find out if a patients endocrine dysfunction appears as the result of primary testicular failure or hypothalamic and/or pituitary deficiency.

Other tests are also recommended. The measurement of serum prolactin is recommended for patients with signs and symptoms of a pituitary tumor, patients whose serum testosterone level is low and whose do not have elevated serum LH that does not have any connection with it and for patients consuming psychotropic drugs or centrally acting antihypertensives.

An evaluation of other pituitary hormones (adrenocorticotropic hormone [ACTH], thyroid-stimulating hormone [TSH], and growth hormone [GH]) is required for all patients who have hypogonadotropic hypogonadism (LH and FSH deficiency).

2. Additional Laboratory Tests
Between 10% and 20% of infertile couples were evaluated and it was observed that they suffer from “unexplained” infertility. The female infertility has lower data due to techniques held to find out the effectiveness of evaluation. During the male infertility one should pay attention to sperm number and motility, the determination conducted by routine semen analysis and the definition of function or true sperm quality. That’s why tests are required for the identification of other abnormalities of semen parameters. These tests are leukocyte, tests of sperm function and antisperm antibody identification.

2.1. Quantitation of Leukocytes in Semen
The identification of leukocytes in semen has been conducted with monoclonal antibody technology. Some patients, who suffer from infertility, have a lot of round cells in their semen. One may make a difference between immature germ cells and leukocytes conducted with a standard semen analysis. The cell type should be determined due to pyospermia considered as infection. It was found out that infertile men have higher white blood cell counts in their ejaculates comparing with normal men.

2.2. Antisperm Antibody Testing
There is a connection between antisperm antibodies (ASA) in the semen and lower pregnancy rates. It may be a result of some risk factors, such as previous genital infections, testicular trauma or biopsy, heat-induced testicular damage, or genital tract obstruction. ASA may also be found out after semen analysis with clumping/agglutination, diminished motility, a poor postcoital test, or the availability of the “shaking” phenomenon on sperm-cervical cross-mucus testing.

These antibodies have been detected with various methods, the most accurate analyze is the immunobead test. Its purpose is the utilization of polyacrylamide beads to which rabbit antihuman antibodies have been connected. It makes possible the acute detection of IgA or IgG antibody connected with the head, midpiece, or tail of motile sperm. More than 20%-50% of sperm demonstrating immunobead binding is not important. Tests evaluating antisperm antibodies in the serum or seminal plasma are not as important as sperm-bound antibody analyzes due to the sperm surface antibodies that have an ability to produce the functional deficits that are connected with immunologic infertility.

3. Advanced Sperm Fertility Tests
Single sperm analyze is not the only indicator of male infertility. That’s why the ability of the spermatozoon to fertilize can be evaluated with different tests and it is known that there is no standard advanced sperm function test that can be applied. One should pay attention to the aim for semen measurements and sperm function tests.

3.1. Strict Morphology
One of the main characteristics of the semen analysis is the percentage of spermatozoa. If this percentage was below the norm, such patients were regarded as teratozoospermic. In 1986 “strict” criteria was accepted with a clinically important norm of 14% normal forms. The measurements taken from spermatozoa that migrated to the cervix without any obstacles considered to be important for these criteria. Normal sperm morphology due to criteria is a good predictor of in vitro fertilization (IVF) rates. The IVF rates were decreased very much among patients who have lower than 14% morphologically normal spermatozoa and had been decreasing due to percentage less than 4%.

For the evaluation of sperm morphology spermatozoa should be located in the Papanicolaou stain and analyze should be held on at least 200 spermatozoa per slide. The criteria for the normal spermatozoon are based on a smooth, oval sperm head whose length is 3-5 micrometers and whose width is 2-3 micrometers. The absence of the neck, midpiece, or tail’s defects are required and an acrosome should include 40%-70% of the sperm head. Cytoplasmic droplets should not exceed the size of the sperm head’s half. All borderline forms considered to be abnormal. There are such abnormal head forms as tapered, pyriform, duplicated, macro, micro, and amorphous. Other forms are also possible. Stricter criteria and normal sperm forms of sperm are 30%.

Such simple test has an advantage of IVF rates prediction and IVF rates optimization by making the concentration of inseminated spermatozoa for teratozoospermic patients higher. Recently, strict morphology was taken as a routine laboratory sperm test.

3.2. Computer-Assisted Semen Analysis (CASA)
CASA was conducted in the 1980s as an automated, objective, and standardized evaluation of sperm concentration and movement. During the analyze sperm density, percent motility, straight-line velocity, curvilinear velocity, linearity, average path velocity, amplitude of lateral head displacement, flagellar beat frequency, and hyperactivation were detested. Digitalized sperm images visualized by a video camera and analyzed by a computer were the main characteristics necessary for the analysis.

Standardization of specimen preparation, cost, technician expertise, and an understanding of the limitations of computer-based analysis being understood are disadvantages of CASA. Moreover, some problems may occur with the accuracy during CASA due to measurement of spermatozoa at very high or very low concentrations. CASA applied in the andrology laboratory can not be always used due to problems connected with understanding of the specifications, the lack of equipment and due to little clinical advantage of CASA comparing with routine semen analysis.

3.3. Hypo-Osmotic Swelling Test (HOS)
In 1984 it was found out that under hypo-osmotic conditions (150 mOsm/L), a normal spermatozoon will absorb fluid that is available after bulging of the plasma membrane and curling of its tail. The principle that a living spermatozoon can maintain an osmotic gradient and a dead cell cannot have such ability was a dominant principle of test.

Phase-contrast microscopy can detect this curling. The purpose of the test is the measurement of the physical and functional integrity of the plasma membrane and as its result viability. It was observed that less than 50% of spermatozoa swell in an abnormal sample; more than 60% of spermatozoa react in a normal one. The test can give functional information on which fertility tests do not influence. Such information is required when no swelling is observed; it was connected with bad IVF results. This analysis helps to hold a differentiation between immotile but viable spermatozoa and necrospermia. The purpose of HOS test is to choose live testicular sperm intracytoplasmic sperm injection (ICSI) in the case of its little quantity and the absence of motility.

3.4. Viability Stain Assays
The purpose of viability stains are to find out if spermatozoa are alive and if the plasma membrane is intact. During these tests the results are held on one principle such as live cells have an ability to exclude dye and damaged dead do not have such ability. The stains that are applied are eosin Y and trypan blue. There is a close connection between vitality results of the stain analysis and the HOS test. Their purpose is the evaluation of the plasma membrane’s integrity. This analysis does not have an ability to predict prognosis of IVF results and put a diagnosis. Very low or absent motility (done from necrospermia) are cases, when diagnosis can not be put. When sperm are stained, they are lifeless and cannot be applied for ICSI.

3.5. Cervical Mucus/Sperm Interaction Assays
The way of spermatozoa can be through the cervical mucus reach to the uterus. Spermatozoa that cannot go through the cervical mucus is the primary cause of infertility found in 10% of couples after a visit to a doctor. The quality of the cervical mucus depends on the menstrual cycle.

The postcoital test (PCT), first conducted by Sims more than 125 years ago, has been used for the determination of cervical mucus/sperm interaction. The purpose of the test is to evaluate sperm concentration and motility in an aspirate of cervical mucus at midcycle soon after an intercourse. When PCT does not have any questions connected with its results, 20 or more spermatozoa are available per high-power field. An abnormal PCT results are connected with inappropriate timing of coitus. ASA, anovulation, an abnormal hormonal milieu, female or male genital tract infections, poor semen quality, and male sexual dysfunction can also lead to abnormal PCT results.

These tests are held beyond the control of the clinic and may consider being useless. The availability of motile spermatozoa is necessary to find out if spermatozoa can survive in the cervical mucus.
Tests should be conducted in hospital for the investigation of the in vitro interaction between spermatozoa available in semen and sperm-free midcycle mucus. The purpose of In vitro tests and the capillary test is to standardize the sperm-mucus penetration ability. The crossed mucus-hostility analysis that demands the apply of donor spermatozoa and the control of mucus, is used to find out who, the male or female partner the cause of poor sperm-cervical mucus interaction. Bovine cervical mucus similar to human cervical mucus both biochemically and physiologically is used in a commercial analysis. Such analysis does not evaluate the female constituent of the cervical factor. The Tru-Trax analysis (Humagen) unites biochemical and physiological approaches, putting human and bovine cervical mucus in adjacent wells. A bad connection of cervical mucus penetration analysis with IVF and pregnancy rates were observed, other people do not accept it. These analyses are not connected with one another, with motility and other semen variables. The purpose of cervical mucus penetration test is to measure a sperm function independent of other sperm functions being measured.

3.6. Sperm Capacitation Assays, Mannose-Ligand Receptor Assays, and Acrosome Reaction Assays
The purpose of these tests is research aims.