Tests & Procedures
Evaluating Male Fertility
Many advances have been made in the ability to diagnose and treat fertility problems. It is essential that a complete and accurate diagnosis be made to determine the appropriate course of therapy. The Andrology laboratory offers several diagnostic tests, including semen analysis, sperm penetration assay and anti-sperm antibody test.
Acrosome Stimulation Test
The acrosome cap is a structure within the sperm head surrounding the upper 40 to 60% of the sperm nucleus. The proper function of the acrosome is required for the sperm to penetrate the egg coating, or zona pellucida. At completion of the acrosome reaction, binding sites on the sperm that are required for sperm/egg fusion are exposed. The acrosome stimulation test uses a double-detection system, which identifies viable sperm that have acrosome reacted following an artificial stimulation protocol.
Anti-Sperm Antibody Test
In males, barriers exist to "hide" sperm from the body’s immune system. This is to keep the body from identifying the sperm as foreign, classifying it as dangerous and producing a defense against it. When these barriers break down the body produces anti-sperm antibodies. If these antibodies attach themselves to sperm they cause severely diminished motility and/or agglutination. Females may also produce anti-sperm antibodies. The presence of antibodies is evaluated by testing seminal fluid, semen and the serum from the woman. There should be an anti-sperm antibody evaluation if: in preparation of a vasectomy reversal, sperm motility is diminished, there is an increase of agglutination observed or if sperm viability is in question. Females should be evaluated if a sperm cervical mucus interaction assay test necessitates it.
Cervical Mucus Interaction
Ovulatory cervical mucus along with the patient’s sperm and fertile donor sperm are placed side by side on a glass slide and cover slipped. The motility of sperm that have penetrated the mucus is recorded at 30 minute intervals. This test will help determine the ability of sperm to penetrate ovulatory mucus. If the fertile donor sample performs better that the patient’s sperm, then there may be male factor infertility. If both sperm samples are impaired, the cervical mucus may be creating a hostile environment.
Hamster Egg Penetration Test
The Hamster Egg Penetration Test (HEPT) (also know as the Sperm Penetration Assay) is the most accurate test in predicting fertilizing ability, and can also predict the ability of laboratory techniques to improve sperm fertilizing ability. The sperm samples are evaluated in much the same manner as with in vitro fertilization (IVF), except that the eggs (chemically treated to allow normal human sperm to penetrate them) are obtained from a hamster.
The prepared sperm are incubated with 15-20 chemically treated eggs. If functionally competent the human sperm can complete the first steps of fertilization including the penetration of the egg, but nothing happens beyond that point. The penetrated eggs are counted and a percentage is calculated.
When test results are compared; if less than 50% of the eggs are penetrated in the non-stimulated sperm, there is a decreased ability to fertilize. A score higher than 50% indicates that the sperm should have the ability to fertilize.
If no sperm are found in the semen or if the count is extremely low, a blood sample may be obtained for the purpose of evaluating the levels of FSH, LH, free testosterone and total testosterone and prolactin. Clues from these tests may lead to probable causes and appropriate therapies.
Krueger or “Strict Criteria” Morphology
The term strict criteria morphology analysis refers to a specific grading technique that evaluates the shape of the sperm. The sperm morphology has been shown to be predictive of the fertilizing ability of the sperm when combined with other measures of fertility. The test includes a very stringent evaluation of the sperm head, tail, mid-piece and tail attachment.
Sperm viability or the percentage of viable sperm may be tested using the Hypoosomatic solution assay. This test examines the sperm’s plasma membrane and its ability to function, which may indicate its fertility. The seminal fluid’s viscosity is evaluated in relation to sperm motility. It is normal for the seminal fluid to have a small number of white blood cells, but an increased presence of these cells may indicate an infection or prostatitis (inflammation of the prostate). If the white blood cell count necessitates a semen culture, a diagnosis and treatment for the problem may be found.
Five to ten percent of fertility problems among couples are caused by abnormalities in the cervix and its secretions. In order for sperm to successfully migrate through the cervix, the sperm must be able to penetrate the mucus. The lower cervix helps store sperm and releases it into the upper tract of the cervix before it enters the uterus.
The physical and chemical properties of cervical mucus vary with changes in the menstrual cycle. These changes affect the pH, viscosity, volume, cell content and spinnbarkheit (measurement of stretch ability) in the cervical mucus ideally making it more receptive to sperm penetration just prior to ovulation.
Retrograde Semen Analysis
A retrograde semen analysis is indicated for patients with a low volume and a diminished sperm count in the initial semen analysis. Retrograde ejaculation is the ejaculation of sperm into the bladder. Urine released following sexual activity will contain sperm in men who undergo retrograde ejaculation. Many retrograde ejaculation patients will have had prior surgery or a medical condition that predisposes them to retrograde ejaculation. Predisposing factors include testicular cancer surgery (RPLND), transurethral surgery of the prostate, or childhood bladder surgery. Medical conditions such as diabetes, MS, or spinal cord injury may also predispose an individual to retrograde ejaculation.
The Semen Analysis is the most efficient and economical method of evaluating male fertility. It is usually the "starting point" for the evaluation of male infertility. The evaluation of the sperm includes: sperm concentration, progressively motile sperm count and sperm morphology (shape).
Decreases in sperm concentration and sperm motility affect the motile sperm count, which reflects the ability to get a high enough concentration of sperm to the egg site to complete fertilization. Sperm morphology is important because it too reflects upon the ability of the sperm to fertilize an egg. A normal semen sample may consist of 35% normal, correctly shaped sperm and the remaining 65% may be abnormally shaped or abnormal sperm. A smaller percentage of normal sperm could result in reduced fertility.
Semen cultures are performed when there is an increase in white blood cells present in the semen sample or upon the request of the referring physician. The semen sample is cultured for a bacterial growth on specialized media. Samples exhibiting significant growth are sent to a referral lab for identification of the bacteria and what antibiotics can kill the bacteria. More information on the presence of white blood cells in semen is available upon request.
Sperm Aneuploidy by FISH
FISH (Fluorescent in-situ hybridization) allows the identification and count of chromosomes as well as any structural defects such as chromosome translocations. By analyzing sperm with FISH prior to an IVF cycle or artificial insemination procedure, it is possible for the care provider to better counsel their patients as to the possible risks during infertility treatment.
Sperm Chromatin Integrity Test (SCIT)
SCIT assess chromatin integrity and DNA strand breaks, which have been shown to correlate with embryo quality. DNA breaks are normally found during chromatin remodeling, however, this damage is repaired during the late stages of spermatogenesis. Retention of sperm DNA breaks may affect fertilization ability and embryo quality.
The sample is divided into two aliquots. One fraction will be used to evaluate DNA damage in the fresh sample, and the second portion will undergo the density gradient centrifugation and DNA damage will be analyzed in the final preparation.
The length of time sperm are able to remain viable after ejaculation may be of concern in some cases. The sperm longevity test can asses the viability of the sperm at given intervals after having been washed free from the seminal plasma and stored in a solution optimized for sperm viability.
Transmission Electron Microscopy
The diagnosis of many sperm pathologies requires the use of transmission electron microscopy. The electron microscope uses very thinly sectioned sperm embedded in a special plastic to look at the sperm’s sub-cellular components at high magnification. The sperm DNA acrosome, tail structure, and membrane structure.
Varicoceles are swollen veins within the scrotum. They are found in fertile as well as infertile men and are extremely common, but since the incidence of their occurrence is greater among infertile men they are believed to be harmful to fertility. They are associated with a decreased number of normal sperm and an increase of "tapered" or abnormal sperm. It also seems to be associated with lower sperm concentration and motility. Varicoceles are suspected to affect capacitation. As with other forms of infertility, it is not associated with complete sterility, but rather with reduced fertility.
If semen analysis results are consistent with varicocele-like semen the patient may be referred to a urologist who will evaluate if a varicocele is present (usually directly above the testicle) and determine the proper treatment. There may be no need for treatment depending upon the degree of affect the varicocele may be having or upon the plans of the couple to have more children. A surgical procedure called a varicocelectomy may be performed on an outpatient basis to correct the problem.
If no sperm are found in the seminal fluid an evaluation of fructose will be performed. A lack of fructose may indicate a blockage of the vas deferens, which secretes fructose and carriers the sperm from the epididymis to the prostate gland.
X-Y Sperm Identification by FISH
Identifying the number of X or Y chromosome bearing sperm in an ejaculate can be beneficial to couples seeking treatment. This can be evaluated in a highly accurate manner when done by directly counting the number of sperm carrying either the X chromosome, those sperm that would result in a female child, or the Y chromosome, those sperm that would result in a male child.