Assisted Reproductive Technology: Laboratory Aspects

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Chapter 39 Assisted Reproductive Technology: Laboratory Aspects


Assessment of semen quality is a critical first step in the evaluation of the infertile couple. The laboratory evaluation of semen quality involves the assessment of sperm fertilizing ability. However, there are many limitations of current laboratory procedures for the assessment of this sperm function. Nonetheless, an expectation of the probability of fertilization is important for the determination of the specific ART procedure required.

Another important laboratory step is the preparation of sperm or sperm-containing tissue for fertilization of oocytes.

Semen Collection

Evaluation of infertility is the most common reason for basic semen analysis. Another common medical indication for this procedure is follow-up after a vasectomy to confirm sterility. Men with threatened future fertility may have their semen analyzed before storage for future insemination.

Regardless of the reason for semen analysis, it is important to realize that the production and submission of a specimen of semen for analysis may be difficult, embarrassing, or distasteful for some patients. For patients undergoing infertility evaluation, there may be associated feelings of frustration, which may make the collection of a semen sample a particularly stressful or unpleasant event. For these reasons, it is vital to have experienced staff to adequately prepare the patient for semen collection. Written and oral instructions should be given well in advance of planned specimen production. The patient should be counseled to abstain from sexual activity for a period of 2 to 5 days before semen analysis. This period should remain consistent within patients to reduce intersample variation with repeated measures.

Many clinicians limit male infertility evaluation to a single semen analysis if the initial sample is found to be normal. However, a thorough infertility evaluation requires analysis of two ejaculates collected as least 1 month apart because semen quality varies over time.1 If either one of the two ejaculates are abnormal, additional samples should be evaluated. Some clinicians recommend that all fertility patients undergo semen analysis once or twice yearly during the duration of their treatment. More frequent analysis may be indicated in postsurgical patients as a part of routine follow-up.

Masturbation is the preferred method for the production of a complete and uncontaminated semen specimen. The glans penis should be cleaned with a wet towel, avoiding the use of soap. Oil-based lubricants should also be avoided because many are toxic to spermatozoa. In instances when the patient objects to masturbation, coitus interruptus using special condoms designed for semen collection may be utilized. Generally, coitus interruptus should be avoided because vaginal secretions may contaminate the specimen and it is difficult to ensure complete collection. Conventional latex condoms should be avoided because near-ubiquitous spermicidals quickly decrease spermatozoa viability. Other methods of sperm collection will be discussed later.

It is preferable for samples to be collected near the andrology assessment area. Rooms designated for production of semen samples should be located in a quiet part of the facility and doors should be labeled with DO NOT DISTURB signs. Reading materials and/or video aids should be available for those men requiring them.

The patient should be provided with clean, appropriately labeled, wide-mouthed containers for collection. The container should be composed of nontoxic plastic and should be sterile. Containers obtained by the patient at home may harbor residual detergents or soaps that are toxic to spermatozoa.

Prompt transport of the sample to the laboratory, within 1 hour, is crucial to accurate testing. During this time, the sample should be protected from temperature extremes. Attempts should be made to keep the sample at body temperature during transit. Prolonged exposure to direct sunlight should also be avoided. All samples should be handled before, during, and after analysis using universal precautions.

Semen Parameters

Spermatozoa Concentration

Measurement of the number of spermatozoa in seminal plasma is a critical component of basic semen analysis. Spermatozoa concentration refers to the number of spermatozoa per unit volume of seminal fluid. This is generally expressed in terms of million of spermatozoa per milliliter (million/mL). Spermatozoa concentration alone does not provide a measure of total number of spermatozoa in the ejaculate. This value, the spermatozoa count, is calculated as the product of spermatozoa concentration and ejaculate volume.

There are two well-established techniques for determining semen concentration, both procedures entailing the counting of an aliquot of liquefied semen in a specialized chamber. Regardless of the method utilized, it is imperative to ensure that the semen specimen is well mixed. Gentle agitation of the container is sufficient to ensure good mixing. Vigorous shaking or vortexing should be avoided.

The concentration of spermatozoa in a semen sample can be measured using the Neubauer hemocytometer method. The Neubauer hemocytometer is one of several specialized counting chambers currently in use. Using a positive displacement pipette, both chambers are filled with well-mixed, diluted semen. The chamber is allowed to stand for 5 minutes to allow the cells to sediment. Only distinct spermatozoa within the grid are counted. If isolated heads, tails, or other germinal cells are noted, they should not be counted. After counting one side of the chamber, the second grid should be counted. The two estimates should be averaged; if each count is not within 5% of the average, both should be discarded and another hemocytometer prepared. If both counts are within 5% of their average, the average is divided by the surface area counted and multiplied by the dilution factor to yield the spermatozoa concentration in the original semen sample (millions/mL). The spermatozoa count may then be calculated by multiplying spermatozoa concentration and ejaculate volume.

The Makler counting chamber (Sefi Medical Instruments, Haifa, Israel) is a specialized instrument designed for rapid semen analysis. Since its inception in 1980, the Makler counting chamber has gained wide acceptance and is used in many laboratories. The main advantage of the Makler counting chamber over the Neubauer hemocytometer is that except in cases of extremely high spermatozoa concentration, no dilution of the semen specimen is needed. Additionally, motility and forward progression may be assessed at the time of spermatozoa concentration determination. Although the Makler Chamber simplifies determination of spermatozoa concentration in semen analysis, the method lacks the accuracy of traditional hemocytometry and may be prone to errors.6

The suffix –zoospermia refers to the presence of spermatozoa in the ejaculate. Patients with concentrations less than 20 ′ 106/mL are classified as oligozoospermic. Men with no spermatozoa present in the ejaculate are deemed azoospermic. Azoospermia is diagnosed only after microscopic confirmation of the absence of spermatozoa in the centrifuged pellet of an undiluted sample of semen.

Spermatozoa Motility and Forward Progression

Although the total number of moving spermatozoa is an important indicator of fertility potential, the quality of the spermatozoa movement must also be assessed. As such, both quantitative and qualitative measures of spermatozoa motility are routinely recorded. Motility and progression assessment of the spermatozoa should be performed within 1 to 2 hours of production, with care taken to keep the sample at room or body temperature to avoid decreases in spermatozoa motility.

For quantitative assessment, at least 200 spermatozoa in five separate viewing fields are classified as exhibiting normal movement, abnormal movement, or no movement. Spermatozoa motility is calculated as the percentage of total spermatozoa that demonstrate flagellar motion. In addition to quantitative assessment, several rating systems are used to evaluate the progressive character of spermatozoa movement. Some rank the progression of individual spermatozoa and count them separately; others hold progression as a modal component of motile spermatozoa.

The World Health Organization (WHO) 1999 progression system classifies spermatozoa into one of four categories: A represents spermatozoa with rapid progressive motility; B, slow or sluggish progressive motility; C, nonprogressive motility; and D, no motility.7 The percentage of spermatozoa in each category is reported. A normal semen specimen contains at least 50% motile spermatozoa with the majority exhibiting good (modal progression class >2, WHO class A and B) forward progression. Athenozoospermia is defined as less than 50% of spermatozoa with good motility.

Spermatozoa Morphology

Assessment of spermatozoa morphology provides a sensitive indicator of the quality of spermatogenesis and fertility potential. Many structural abnormalities affecting spermatozoa are associated with infertility. Accurate morphologic characterization of the semen specimen is an important part of the semen analysis. Multiple systems of categorization of spermatozoa morphology are currently in use. Many systems are plagued by subjective interpretations of abnormality, which vary from observer to observer. Newer methods have attempted to standardize morphologic evaluation.

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.

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.

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 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.

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.

Processing of Spermatozoa Collected Following Vibratory Stimulation/Electroejaculation

Failure of emission is termed anejaculation. Causes of anejaculation are similar to those of retrograde ejaculation, although spinal cord injury is a much more common cause. Because most of these patients do not respond to sympathomimetic therapy, they may require the use of external stimulatory devices to achieve emission. The most common methods of stimulation are penile vibratory stimulation and rectal electroejaculation. Penile vibratory stimulation entails the application of a penile vibrator to the glans penis, leading to a reflexive ejaculation. This technique induces ejaculation in approximately 70% of anejaculatory men with spinal cord injuries.17

Men with lesions above the level of T10 are more likely to respond than those with lesions below that level. Electroejaculation may be used in men who do not respond to vibratory stimulation. This procedure involves carefully inserting a probe into the rectum, followed by the intermittent application of electrical current to the periprostatic nerve plexus. The current stimulates the muscular tissue of the prostate, seminal vesicle, vas deferens, and epididymis to contract and propel semen into the urethra. It is successful in approximately 75% of patients.18,19 If antegrade ejaculation ensues, it may be collected from the urethra. If retrograde ejaculation occurs, then the urine is alkalinized beforehand and the bladder irrigated with medium and catheterized to remove any spermatozoa-bearing fluid.

Risks of electroejaculation include rectal thermal injury or perforation. The rectum should be examined with proctoscopy before and after the procedure. Life-threatening autonomic dysreflexia, or massive, uncoordinated sympathetic release, may occur in patients with spinal cord injuries above the level of T6. Blood pressure monitoring is imperative in this patient population. Semen obtained by vibratory stimulation and electroejaculation frequently has low volume, high spermatozoa concentration, and poor motility, likely owing to stasis within the genital tract.20 Again, processing of samples produced with vibratory stimulation or electroejaculation continues as in samples produced with masturbation.