In 1986, Kruger and associates proposed a strict criteria for classification of normal and abnormal spermatozoa morphology.⁸ Objective measurements of individual spermatozoa components are included in this system, also known as the Tygerberg strict criteria classification system. According to this system, a spermatozoon is considered normal only if: (1) the head measures 5 to 6 microns in length and 2.5 to 3.5 microns in width; (2) acrosomal staining covers 40% to 70% of the anterior aspect of the sperm head; (3) the midpiece is 1.5 times as long as the head length and less than 1 micron in width; (4) the tail is approximately 45 microns long, uniform, uncoiled, and free from kinks; and (5) cytoplasmic droplets are no more than half the size of the spermatozoa head and occupy the midpiece only. Borderline forms are considered abnormal in this classification scheme.

In men with spermatozoa counts greater than 20 million/mL and motility greater than 30%, fertilization rates during IVF cycles were 91% for men with greater than 14% normal spermatozoa by Tygerberg strict criteria and 37% for men with less than 14% normal forms.⁸ This study established the reference value for normal morphology as 15% or greater by strict criteria.

In 1992, the WHO adopted a grading system based on the Tygerberg strict characterization of distinct spermatozoon components.³ Under these guidelines, a spermatozoon is considered normal if: (1) the head measures 4 to 5.5 microns in length and 2.5 to 3.5 microns in width; (2) acrosomal staining covers 40% to 70% of the anterior aspect of the sperm head; (3) there are no neck, midpiece, or tail defects; and (4) cytoplasmic droplets are less than one third the size of the head. Although these parameters appear quite similar to Tygerberg criteria, slightly looser standards increase the reference value for normal morphology to 30%. Use of both grading schemes varies from laboratory to laboratory.

Spermatozoa Viability

Spermatozoa viability is tested by evaluating the capacity for the spermatozoa membrane to exclude dyes or other substances. Because the percentage of viable spermatozoa is usually slightly greater than that of motile spermatozoa, viability testing is usually performed only when motility is less than 40%.

The eosin–nigrosin test is a common method for assessing viability. Intact cells exclude eosin dye (red); nonviable cells exhibit red staining. Nigrosin functions as a counterstain to visualize unstained live cells. Equal volumes of liquefied semen and eosin–nigrosin stain are mixed on a clean, dry microscope slide. The total number of spermatozoa that do not take up the stain is recorded as percent of viable spermatozoa in the sample.

Another method for assessing spermatozoa viability is the hypo-osmotic swelling test. The test relies on the ability of live spermatozoa to tolerate moderate hypo-osmotic stress as evidenced by controlled swelling. Dead spermatozoa do not show discernible swelling when exposed to hypo-osmotic conditions, whereas viable spermatozoa exhibit a characteristic coiling pattern in the tail.⁹

Functional Spermatozoa Tests

Despite normal semen parameters, spermatozoa may be functionally unable to perform all the requisite steps necessary for fertilization. Several tests have been developed to evaluate potential functional obstructions to fertility. The predictive value of these tests is questionable, however, and their usefulness in therapeutic fertility treatment is debatable, at best.

Acrosome Reaction

Before fertilization, spermatozoa must undergo the acrosome reaction. The acrosome reaction involves the fusion of the acrosomal membrane with the adjacent plasma membrane. This fusion results in the release of acrosomal content. Evaluation of acrosome functional activity may be accomplished by mixing capacitated spermatozoa with inducers of the acrosome reaction. Spermatozoa are then evaluated using fluorescent stain to determine the status of the acrosomal membrane.

Zona Pellucida Binding Assay

The zona pellucida binding assay is another adjunctive test of spermatozoa function. This test assesses the capacity of spermatozoa to bind to the outer surface of the zona pellucida, a prerequisite for binding and penetrating the egg. Microscopically, the zona pellucida is divided and each half is mixed with a sample of the patient’s spermatozoa or a sample of a fertile donor. Spermatozoa bound to the zona pellucida outer surface are counted. A hemizona index is calculated by dividing the total number of patient spermatozoa bound by the total number of donor spermatozoa bound. Successful IVF is associated with an index greater than 60%.¹⁰

Hamster Egg Penetration Assay

The zona pellucida, the acellular glycoprotein layer surrounding the ovum, regulates species-specific fertilization. Removal of this layer allows evaluation of interspecies, spermatozoa–oocyte interaction using the sperm penetration assay (SPA). This procedure assesses the ability of human spermatozoa to penetrate a hamster oocyte as a test of the fertilizing capability of the human spermatozoa.¹¹ Human spermatozoa penetration of zona-free hamster oocytes has been shown to correlate with human spermatozoa penetration of human ova.¹²

Sperm Chromatin Structure Assay/Single-cell Gel Electrophoresis

Spermatozoa DNA integrity tests are useful in the evaluation of the haploid chromatin of the spermatozoa nucleus. Chromosomal quality is determined by both chromatin maturity and chromatin normalcy. Both the sperm chromatin structure assay (SCSA) and single-cell gel electrophoresis test (also known as comet assay) serve as tools for measuring clinically important properties of spermatozoa nuclear chromatin integrity.

Assays utilize direct staining and evaluation of spermatozoa chromatin to assess the integrity of the genetic material. The tests provide useful information on the presence of genetic damage to spermatozoa DNA. Briefly, both procedures evaluate shifts from intact, double-stranded DNA to denatured, single-stranded DNA.¹³ These assays can be used to assess male spermatozoa DNA integrity as related to fertility potential and embryo development as well as effects of reproductive toxins.¹⁴

Collection-Specific Spermatozoa Evaluation and Processing

For most healthy men, masturbation is a straightforward method of semen collection. For men with ejaculatory dysfunction or azoospermia, however, other methods of spermatozoa recovery are necessary for fertility evaluation or therapeutic maneuvers. Men who are unable to ejaculate may require clinical spermatozoa collection; men with azoospermia may be aided by surgical intervention.

Processing of Semen Collected by Masturbation

Exposure to seminal plasma results in decreased spermatozoa motility and viability. Prolonged exposure can irrevocably diminish the fertilizing potential of spermatozoa. To obtain the heartiest sample for both laboratory tests of spermatozoa function and clinical application, spermatozoa must be separated from the seminal plasma after ejaculation. There are three fundamental approaches to the separation of spermatozoa from seminal plasma: spermatozoa washing; spermatozoa migration, or swim-up; and spermatozoa density gradient separation. These general spermatozoa processing techniques are applicable to both intrauterine insemination (IUI) and IVF procedures.

Spermatozoa Washing

Spermatozoa washing is the most common method for the separation of spermatozoa from seminal plasma. Washing not only quickly and efficiently removes detrimental factors present within the seminal plasma, but also concentrates spermatozoa within the ejaculate. Spermatozoa washing is accomplished by mixing the ejaculate with an appropriate, protein-supplemented medium. Mixtures are then centrifuged and spermatozoa pellets form at the bottom of the container. The supernatant is carefully aspirated and the pellet is resuspended. This procedure can be recommended for samples with good concentrations of highly motile spermatozoa and very few nonspermatozoa contaminating cells.

Spermatozoa Migration Techniques (Swim-up Method)

In the female reproductive tract, migration into the periovulatory cervical mucus isolates a population of potentially functional spermatozoa. Although simple washing removes seminal plasma from a semen sample, it does nothing to accomplish this goal. Techniques that enrich the population of motile or morphologically normal spermatozoa from a sample may be utilized for IUI or IVF, and include the classic swim-up procedure. In the spermatozoa swim-up procedure, the innate ability of motile spermatozoa to migrate against gravity is used to enrich for motile spermatozoa.

Two different methods of swim-up separation are commonly used. In the first (swim-up from semen), freshly liquefied semen is split into several fractions and placed in tubes beneath a layer of low-viscosity culture medium. In the second method (swim-up from pellet), fresh semen is first washed and pelleted. The supernatant is removed and medium is gently layered over the pellet. In both methods, motile spermatozoa naturally migrate from the lower layer (fresh semen or pelleted spermatozoa) into the overlying culture medium during incubation at 37°C.

Spermatozoa washing of a sample before enrichment for motile spermatozoa (swim-up from pellet) has been criticized because dead and abnormal spermatozoa present in the original sample remain in the pellet. Aitken and Clarkson showed that the functional fertilizing capacity of motile spermatozoa isolated from such a pellet can be impaired when compared to that of motile spermatozoa isolated before spermatozoa washing and pelleting.¹⁵ The authors propose that the production of oxygen free radicals by abnormal spermatozoa in the pellet may result in cellular damage in motile spermatozoa, leading to abnormal spermatozoa function. Additionally, the centrifuged pellet has a relatively small interface with the overlying layer, making it difficult for motile spermatozoa in the bottom of the pellet to migrate into the media.

Density Gradient Separation

Density gradient separation relies on a density gradient coupled with centrifugation to separate fractions of spermatozoa based on motility, size, and density. Various techniques have been described using continuous or discontinuous (discrete layers interrupted by defined interfaces) gradients. A variety of commercially available gradient materials are in use. An aliquot of the spermatozoa sample is gently layered over gradients of such a material and centrifuged. The resulting pellet is washed in culture medium before resuspension in medium at the desired concentration. Additionally, one can perform density gradient separation and washing followed by spermatozoa swim-up in bicarbonate-buffered media at 37°C in a CO₂ incubator to select for a highly motile population of spermatozoa for insemination in IVF.

Processing of Spermatozoa Collected Following Retrograde Ejaculation

Retrograde ejaculation involves the flow of semen into the bladder during orgasm. During normal ejaculation, the urinary sphincter remains closed to guard against retrograde expulsion of semen into the bladder. If the sphincter is incompetent or otherwise nonfunctioning, semen may preferentially travel back into the bladder rather than through the urethra. Retrograde ejaculation is a rare cause of male infertility. It should be suspected in patients with oligospermia or aspermia. Any condition that interferes with the sympathetic innervation of the bladder neck or vas deferens may result in retrograde flow. The condition is diagnosed by examining postejaculate urine for spermatozoa. The presence of 10 to 15 spermatozoa/HPF confirms the diagnosis and differentiates retrograde ejaculation from anejaculation.

Viable spermatozoa may be retrieved from the urine of patients failing medical therapy.¹⁶ Because urine’s acidity makes it naturally toxic to spermatozoa, it is first suggested to alter the chemical milieu of the bladder to minimize its deleterious effects on spermatozoa quality. Alkalinization of the urine above a pH of 7.5 can be achieved by instructing the patient to ingest sodium bicarbonate for several days prior to collection. The patient empties the bladder before masturbation. Immediately after orgasm, the patient urinates into a sterile container, which is taken to the laboratory for spermatozoa extraction. Alternatively, for men unable to volitionally void, the bladder may be catheterized and washed with fresh, pH-adjusted, spermatozoa processing medium. After irrigation, a small volume of medium is left in the bladder. The patient masturbates and ejaculates, and the bladder is catheterized again to obtain fluid containing retrograde ejaculate, which is collected and transported to the laboratory for processing. Laboratory procedures begin with centrifugation and removal of urine from the spermatozoa. The resuspended pellet can then be processed as previously described for samples produced from masturbation.