Male Factor Testing

Problems of male fertility include low sperm count (oligozoospermia) or the production of sperm which lack the motility or other critical factors necessary to achieve fertilization. The andrology laboratory of the Loma Linda University Center or Fertility and In Vitro Fertilization provides many tests for evaluating male infertility patients including computerized semen analysis, sperm antibody testing, and sperm penetration assays (hamster test). The Center also provides short-term sperm freezing technologies for artificial insemination using husband or donor semen. Various treatment options include medication, surgery, intrauterine insemination of concentrated sperm, or in vitro fertilization with or without intracytoplasmic sperm injection (ICSI).

The sperm penetration assay or hamster test

The SPA (Sperm Penetration Assay) or hamster test is used to evaluate the capacity of the patient sperm to undergo capacitation and the acrosome reaction. Capacitation refers to sperm membrane changes accompanied by increases in sperm metabolism and motility. Acrosome reaction refers to the opening of the sperm head membrane followed by the release of enzymes. These are the two important and necessary changes in the sperm before the sperm can fertilize the egg. However, the SPA does not evaluate sperm binding and zona pellucida penetration. Sperm enhancement procedures such as percoll and test-yolk buffer treatments of the sperm may be utilized in the event that the sperm show no penetration of the egg. In such a case, the test will be carried out over three full days and rechecked. Your physician will use the test results to formulate a processing method which is best for your sperm.

Interpretation of SPA results

The SPA (Sperm Penetration Assay) is performed in our laboratory by determining the percentage of enzyme-treated hamster eggs penetrated by the patient sperm. A proven sperm specimen called a control is tested alongside the patient's sperm as quality assurance. The passing level for the SPA is 10 percent or more eggs penetrated. Sperm treated with enhancement procedures such as the test yolk buffer wash require a passing level of 20 percent or more egg penetration. In vitro fertilization (IVF) patients may require a 20 percent or more egg penetration criterium. The SPA results may help to decide if a higher sperm concentration is necessary for inseminations. About 90 percent of the patients who pass the SPA will fertilize at least one of the spouse's eggs while 35 percent of the patients who fail the SPA will fertilize some eggs. Since semen samples may differ in quality on different occasions, sometimes it is necessary to repeat testing at another time.

The immunobead test (IBT)

The Immunobead Test (IBT) is used to detect the presence of antisperm antibodies in sperm as well as in the blood of the couple. A complete semen analysis is also carries out on the same specimen as part of the testing. The IBT test provides information regarding the severity of the antibodies, the target site of the antibodies (sperm head, midpiece, tail, or tail-tip) and the types (IgA or IgG) present. The testing of the blood (serum) helps to determine if the antibodies originate from the blood circulation or originate locally at the site of the reproductive organs. Your physician may also suggest the testing of cervical mucus for antisperm antibodies to verify an unsatisfactory postcoital test. Antisperm antibodies are produced in the body by the immune system in response to the presence of unrecognized sperm proteins. They are important factors that have been shown to inhibit the fertilization of the egg. Antisperm antibodies on the sperm's tail may impair the entry of the sperm through cervical mucus into the reproductive tubes and are associated with recurrent, spontaneous abortions.

Interpretation of IBT results

The World Health Organization considers 20 percent or more sperm bound with antibodies as positive, while some IVF centers consider 20 percent or more as problematic for the IVF procedure. The levels become clinically significant at the 50 percent or higher level. Some studies indicate that washing of the sperm may help reduce the number of antibodies binding. Other methods to reduce antibodies binding include steroid therapy, collecting split ejaculate, short abstinence periods, and Sephadex, Imunobead, or Protease processing. These methods will work only in some patients. When antisperm antibodies are present in the patient, our physicians will use the test results to formulate the best processing method for your sperm.

Semen analysis interpretation (Reference in parentheses)

Abstinence (2-7 days): An abstinence period of less than 2 days may show a low sperm concentration (count). Longer abstinence periods, over 5 days, may be linked to decrease sperm enzyme (acrosin) activity or may lower sperm motility due to necrospermia.
Volume (2-6 mL): A low volume or hypospermia suggests obstruction in the duct, retrograde ejaculation, spermatemphraxis, FSH/LH imbalance or an incomplete specimen. Excessive semen volume is considered hyperspermia and this condition may dilute out sperm cells, backwash sperm away or dilute important sperm maturation factors.
Viscosity (normal): A hyperviscid specimen may indicate infection or presence of antibodies.
pH (7.2-7.8): A pH value not in the reference range suggests an infection.
Count (20-250 million/ml): Low or zero counts may be due to FSH/LH inadequacies, blocked ducts, drugs, steroids, vasectomy, congenital anomalies or season (summer). A seminal fructose test helps distinguish obstruction cases. Analyses should be performed on 2 separate occasions to diagnose oligozoospermia. Genetic testing (eg. for cystic fibrosis) and counseling may be considered for men with low or zero counts before the intracytoplasmic sperm injection (ICSI) procedure. High counts may accompany poor sperm motility. Polyzoospermia (> 250 million sperm/ml) may be linked to decreased sperm enzyme (acrosin) activity and/or miscarriages. Clumping of sperm or debris may cause inaccuracies in the counting of sperm.
Viability (>75 percent): This parameter shows what is the percentage of live sperm in a specimen. Immotile sperm appearing dead (eg. after vasectomy reversal, fructose deprivation, antibodies) may be shown to be alive by this parameter.
HOS Hypoosmotic swelling (>60 percent): The HOS measures the integrity of the sperm TAIL membrane and detects leaky tail membranes.
VHOS Viability in hypoosmotic solution (>40 percent): This is a new parameter first reported by the Loma Linda University IVF group which provides information on the integrity of the sperm head membranes. Low values may be associated with male factor infertility. Gentle sperm washes may be used on sperm with low VHOS values. Low VHOS and HOS together may equal poor sperm fertilizability.
Rapid progressive motility (>25 percent): This is equal to sperm with index 4. The rapid progressive category has been correlated to the fertilizing power of the sperm. Rapid progressive sperm are usually the vanguard sperm. A cold specimen may have few rapid progressive sperm.
Total progressive motility (>50 percent): This category may indicate the overall "health" of the specimen. Low total motility may also occur if energy substrates eg. fructose, are inadequate or if seminal lipid levels are excessively high. Total loss of motility also may be due to Kartegener, Usher, X-linked pK-A AKAP82 or autosomal dominant polycystic kidney disease.
Hyperactivation: Motile sperm may show high amplitude, thrashing or star spin "dance" movements called hyperactivation. Percent hyperactive sperm may predict the fertilizing ability of the sperm population, in particular, for in vitro fertilization patients.
Computer assisted sperm motility analysis (CASA): CASA parameters such as curvilinear velocity combine to characterize the motility pattern which may predict sperm fertilizability. Research studies are still in progress on CASA.
Acrosome Intact (>16 percent): Low percentages of sperm with intact acrosome suggest low fertilization.
Round cells (Neutrophils < 1 million/ml): High numbers of neutrophils may signal an infection.

Comments on possible interpretations of sperm morphology

Strict Criteria Normal Morphology (>4 percent): The normal morphology based on the Tygerberg strict criteria system described by Dr. Kruger et al. [Fertil Steril 46,1118,1986] classifies a sperm as normal when the head size and acrosome are within a set range and there are no structural defects. A value of more than 4 percent has been correlated to in vitro fertilization of oocytes. A low value (teratozoospermia) may require the ICSI treatment.

Dag axonemal defect of midpiece and tail: The sperm tails are coiled within a stretched plasma membrane around the head. The sperm is immotile and the axonemal structure is abnormal (disrupted, broken, loss of fibers). The defect is reported to originate in the epididymis and not testis.
Dysplasia of Fibrous Sheath: This is also known as stump-tail sperm defect and the tail structure is shortened with abnormal mitochondrial assembly at the midpiece.
Round-head /head defects: Sperm with this feature called globozoospermia may lack enzymes to penetrate the egg.

Pinhead /head defects: Diabetics may have many pin-head sperm..
Cytoplasmic droplets: This may be due to frequent ejaculation, excessive physical exercise or incomplete spermiogenesis.
Tapered: This category and pinhead (head defects) sperm may be indicative of varicocele.
Duplicates: This may be linked to chemical exposure, metals, smoke or high prolactin.
Neck/Midpiece defects: Damaged midpiece suggests defective centrioles or segmental mitochondrial aplasia.
Coiled-tail /tail defects: This suggests the presence of antisperm antibodies. A high percentage and low or zero motility may indicate Dag axonemal defect.

Reference: World Health Organization. 4th Edition, WHO laboratory manual for the examination of human semen and semen-cervical mucus interaction. Cambridge University Press. Cambridge, 1999

Andrology Lab Section, Loma Linda University, California.

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