Infertility

OVERVIEW
Reproductive medicine has undergone tremendous changes during the 1990s. The concept of cloning humans from cells from a person’s nose as suggested in Woody Allen’s film Bananas may not be as farfetched as it seemed in 1971 when the film was released. The advances in current reproductive technologies were made clear after the cloning of a sheep, resulting in the birth of Dolly (Wilmut et al, 1997). Although cloning humans has not been accomplished and the concept remains highly controversial, there have been steady advances in the assisted reproductive techniques (ARTs), such as blastocyst transfer and embryo biopsy. Sperm may often be recovered from the testes of patients with nonobstructive azoospermia and injected into individual human ova (intracytoplasmic sperm injection [ICSI]). With the success of these high-technologic, high-cost procedures, the evaluation of the male is often bypassed. This approach ignores the fact that many causes of male infertility, such as varicocele, ductal obstruction, and infections, are easily and effectively treated. In addition, without a full evaluation, significant diseases, such as testicular cancer, pituitary tumors, and neurologic disorders, may be overlooked (Jarow, 1994). Finally, as greater inroads into the genetic causes of male infertility have been made, there has been increased importance placed on the proper evaluation and counseling for the male partner before beginning the ARTs.The chance of a normal couple conceiving is estimated to be 20% to 25% per month, 75% by 6 months, and 90% by 1 year (Spira, 1986). Most conceptions occur when intercourse takes place within 6 days before and including the day of ovulation. Few pregnancies occur following intercourse solely after the day of ovulation. The timing of intercourse, relative to ovulation, has no relation to the sex of the child (Wilcox et al, 1995). In addition, fertility rates are at their peak in men and women at age 24 years. Beyond that age, fertility rates begin to decline with age in both sexes (Noord-Zaadstra et al, 1991; Ford et al, 2000).

Studies of couples of unknown fertility status who are attempting to conceive have demonstrated that, although most couples achieve conception within 1 year, approximately 15% of couples are unable to do so (Hull et al, 1985; Greenhall and Vessey, 1990; Thonneau et al, 1991). Approximately 20% of cases of infertility are caused entirely by a male factor, with an additional 30% to 40% of cases involving both male and female factors (Mosher and Pratt, 1991; Thonneau et al, 1991). Therefore, a male factor is present in one half of infertile couples.Of infertile couples without treatment, 25% to 35% will conceive at some time by intercourse alone (Collins et al, 1983). Within the first 2 years, 23% will conceive, whereas an additional 10% will do so within 2 more years (Aafjes et al, 1978). This baseline pregnancy rate of 1% to 3% per month (in nonazoospermic couples) must be kept in mind while managing infertile couples and evaluating the results of therapy. Although, in the past, couples were not evaluated until 12 months of attempted conception, with the advancing age of infertile couples, we do not recommend deferring an initial evaluation. A basic, simple, cost-effective evaluation of both the male and the female should be initiated at the time of presentation.The approach to the evaluation of the infertile male should be similar to that used to evaluate other medical problems. A thorough history should be obtained, with particular attention to those areas that may affect fertility. This should be followed by a physical examination. An initial series of laboratory tests completes the basic evaluation. The results of the history, physical examination, and initial laboratory testing are used to formulate a differential diagnosis that may lead to more specific testing. Many tests are available to evaluate different aspects of male infertility, but not all patients require all tests.

The goals of the evaluation of the infertile male are to identify (1) reversible conditions; (2) irreversible causes that may be managed by ARTs using the male partner’s sperm; (3) irreversible conditions that may not be managed by these techniques and in which the couple should be advised to pursue donor insemination or adoption; (4) significant underlying medical pathology; and (5) genetic and/or chromosomal abnormalities that may affect either the patient or his offspring. Ideally, the evaluation of the infertile man should result in the identification of the specific abnormality responsible for infertility. Although this is possible in some instances, many men have abnormal semen analyses for which no cause can be identified. When possible, specific treatment is directed toward a specific cause. However, both empirical therapies and ARTs, such as intrauterine insemination (IUI) and in vitro fertilization (IVF), may be of value in the absence of known etiologic factors. It is important to remember that therapeutic donor insemination and adoption are treatment alternatives. The infertile couple should be made aware of these options, with the physician playing a counseling role to avoid excessively prolonged, futile treatments.

PHYSICAL EXAMINATION

The physical examination should be directed toward identifying abnormalities that may be associated with infertility. The patient’s habitus as well as the pattern of virilization should be noted. Abnormalities of the secondary sex characteristics may indicate whether there is a congenital endocrine disorder such as a eunuchoid appearance associated with Klinefelter’s syndrome. Lack of temporal pattern balding and fine wrinkles on the face may be indicative of an acquired androgen deficiency. Gynecomastia is suggestive of either an estrogen/androgen imbalance or an excess of prolactin. Situs inversus raises the possibility of Kartagener’s syndrome associated with immotile cilia and thus immotile sperm.

Genital Examination

Specific attention should be directed toward the genital examination. The penis should be examined for evidence of hypospadias and severe chordee. Both of these may interfere with proper deposition of semen in the deep vagina near the cervix. The scrotal contents should be examined with the patient standing in a warm room to allow for relaxation of the cremaster muscle. The testes should be carefully palpated to determine consistency and to rule out the presence of an intratesticular mass. Because the majority of the testicular volume (∼80%) consists of seminiferous tubules and germinal elements, a reduction in the number of these cells is typically manifested by a reduction in testicular volume or testicular atrophy.

The dimensions of the testes should be measured. This may be performed using calipers, an orchidometer, or sonography (Takihara et al, 1983). The normal adult testis is larger than 4 × 3 cm in its greatest dimensions or greater than 20 ml in volume for whites and African Americans ( Table 43–2) (Carney and Tuttle, 1960). Asian men normally have smaller testes. Decreased testicular size, whether unilateral or bilateral, correlates with impaired spermatogenesis (Lipshultz and Corriere, 1977). Careful palpation of the epididymis should determine the presence of the head, body, and tail. The possibility of epididymal obstruction is suggested by the presence of induration or cystic dilatation of the epididymis. Spermatoceles and epididymal cysts are common findings and do not indicate the presence of obstruction. Palpation of the vas deferens is performed to ensure its presence as well as to rule out areas of atrophy.

Examination of the spermatic cords should be performed to identify the presence of a varicocele. Small varicoceles (grade I) are palpable only during the Valsalva maneuver. Moderate-sized varicoceles (grade II) are palpable with the patient in the standing position, and large varicoceles (grade III) are visible through the scrotal skin and are palpable when the patient is in the standing position. Asymmetry of the spermatic cords, accentuated by the Valsalva maneuver, suggests the presence of a varicocele. In patients with strong cremasteric reflexes, or in those with high-riding testicles, slight traction on the testes during the Valsalva maneuver allows for a more accurate examination of the spermatic cords. Thickening and asymmetry of the spermatic cords that persists in the supine position suggests the possibility of a lipoma of the cord or caval obstruction caused by a retroperitoneal or renal tumor. Varicoceles decrease in size when the patient is in the recumbent position. Similarly, bilateral thickening of the cords, resolving with the patient in the supine position, suggests the presence of bilateral varicoceles.Many diagnostic methods have been employed to identify subclinical varicoceles, those that are not palpable by physical examination. Venography has been used for many years and is considered by some to be a “gold standard.” However, venography is invasive and is not without complications (Seyferth et al, 1981). The results of venography are affected by the position of the catheter tip, the pressure of injection, and the judgment of the examiner. The presence of a venous rushing sound that increases with Valsalva maneuver is used to identify the presence of a varicocele with the Doppler stethoscope (Greenberg et al, 1979; World Health Organization, 1985). Both real-time scrotal ultrasonography and duplex scrotal ultrasonography have been used to visualize dilated spermatic veins in the scrotum. The presence of multiple veins with at least one larger than 3 mm in diameter is believed to indicate a subclinical varicocele (McClure and Hricak, 1986; Eskew et al, 1993), whereas a diameter of 3.5 mm by ultrasound is more predictive of a clinical varicocele (Meacham et al, 1994; Jarow et al, 1996). Higher temperatures in the scrotum or testicle that are identified with contact scrotal thermography are believed to correlate with the presence of a varicocele (World Health Organization, 1985; Comhaire, 1991; Trum et al, 1996).However, all of these techniques are too sensitive, and, in the absence of a universally agreed on gold standard diagnostic test, it is difficult to determine their specificity. With the use of these diagnostic methods, up to 91% of patients with idiopathic infertility have been identified as having subclinical varicoceles, bilateral varicoceles have been demonstrated in up to 58% of patients, whereas only 10% of patients are identified as having bilateral varicoceles by clinical examination alone (Perrin et al, 1980; Narayan et al, 1981; Gonzalez et al, 1983). There have been no controlled studies demonstrating improved pregnancy rates after the diagnosis and treatment of subclinical varicoceles. We therefore do not recommend evaluating patients for the presence of subclinical varicoceles.

Finally, many experts recommend that a careful rectal examination should be done to evaluate the prostate as well as the areas above the prostate for evidence of cystic dilatation of the seminal vesicles. Significant prostatic tenderness raises the possibility of a prostatic infection. However, many abnormalities of the prostate and seminal vesicles observed with transrectal ultrasonography are not detected by rectal examination (Jarow, 1993).

INITIAL BASIC LABORATORY EVALUATION

After the history and physical examination, the male partner of an infertile couple should have appropriate laboratory testing performed . All patients should have at least two or three semen analyses.

Semen Analysis

The semen analysis remains the cornerstone of the laboratory evaluation of the infertile man. Despite this, it is important to realize that the measurement of semen parameters does not necessarily constitute a measure of fertility . Except in cases of azoospermia, the semen analysis does not allow for the definitive separation of patients into sterile and fertile groups. As semen parameters decrease in quality, the statistical chance of conception decreases but does not reach zero. Nevertheless, an accurately performed semen analysis remains an important tool for the evaluation of the infertile man.

Collection

To compare different semen samples from the same patient with accuracy, it is important to maintain consistency in the duration of sexual abstinence before collection of the specimen. Changes in the intervals between ejaculations often results in an increased variability of the results of the semen analysis. Even with this precaution, the results of semen analyses are often inconsistent and, therefore, multiple analyses are indicated in equivocal or difficult situations. Clean, wide-mouthed containers should be used for specimen collection. These containers should be obtained from the physician because residual chemicals in other containers may injure sperm. The specimen may be collected in the physician’s office or at home and brought to the office by placing the container in a shirt pocket next to the body to keep it warm during transit.Most specimens are obtained by masturbation. In those cases in which the patient objects to collecting the specimen through masturbation, special condoms designed for semen collection may be used, allowing the couple to have intercourse. Ordinary latex condoms should not be used because they interfere with the viability of sperm and often contain spermicides. Although coitus interruptus may be used as an alternative method for obtaining specimens, this is not an ideal method because the initial portion of the ejaculate may be lost and bacteria and acidic vaginal secretions may contaminate the specimen. The specimen should be examined in the laboratory within 1 to 2 hours of collection. A label on the container should state the patient’s name, the date, the time of collection, and the abstinence period . In most cases, two or three specimens examined over a period of several weeks will give an adequate assessment of baseline spermatogenesis. In those occasional cases in which parameters differ markedly in the initial semen specimens, additional specimens, collected over a 2- to 3-month period, may be obtained.

Methodology

Physical Characteristics

Freshly ejaculated semen is a coagulum that liquefies over a 5- to 25-minute period. The seminal vesicles secrete the substance responsible for coagulation . Patients with CBAVD usually have absent or hypoplastic seminal vesicles. Semen in these patients does not coagulate, is acidic, and has a low volume.Secretions from the testis, epididymis, bulbourethral glands (Cowper’s glands), glands of Littre (periurethral glands), prostate, and seminal vesicles compose the normal seminal fluid. The fluid is released from the glands in a specific sequence during ejaculation. Before the ejaculation of the major portion of the ejaculate, a small amount of fluid from the glands of Littre and the bulbourethral glands is secreted. This is followed by a low-viscosity opalescent fluid from the prostate containing a few sperm. The principal portion of the ejaculate contains the highest concentration of sperm, along with secretions from the testis, epididymis, and vas deferens, as well as some prostatic and seminal vesicle fluids. The last fraction of the ejaculate consists of seminal vesicle secretions. The secretions from Cowper’s glands account for 0.1 to 0.2 ml, prostatic secretions account for 0.5 ml, and the secretions from the seminal vesicles account for 1.5 to 2.0 ml. The majority of ejaculated sperm come from the distal epididymis, with a small contribution from the ampulla of the vas. The unobstructed seminal vesicle is not normally a reservoir for sperm.Semen liquefaction is due to prostatic-derived proteases, including prostate-specific antigen and plasminogen activator. Failure of liquefaction should be differentiated from semen that remains hyperviscous after liquefaction. Nonliquefied semen remains a coagulum and does not change consistency after ejaculation. Hyperviscous liquefied semen becomes less of a coagulum after ejaculation; however, its consistency remains thicker than normal. Although tissue plasminogen activator levels have been shown to be lower in the semen of patients with nonliquefaction than in normal semen (Arnaud et al, 1994), that is not the case with prostate-specific antigen levels (Dube et al, 1989). The effect that semen nonliquefaction has on male fertility remains unclear. Some patients with nonliquefying semen have normal postcoital test (PCT) results. In addition, sperm may be found in the cervical mucus before semen liquefaction (Santomauro et al, 1972). Although liquefaction of semen may be induced by the addition of seminin (a seminal protease) or α-amylase, there is no evidence that treatments with these agents increase fertility (Syner et al, 1975; Wilson and Bunge, 1975). Liquefied semen should be able to be poured drop by drop, whereas hyperviscous semen forms thick strands instead of drops.The cause of semen hyperviscosity remains controversial. Although, in the past, semen hyperviscosity has been thought to be due to genital tract infection, recent studies do not support this (Munuce et al, 1999). Similarly, seminal protein patterns appear similar between normal and hyperviscous semen specimens (Carpino and Siliciano, 1998). There is some evidence that dysfunction of the seminal vesicles may be involved in semen hyperviscosity, because seminal fructose levels are lower in those samples with semen hyperviscosity (Gonzales et al, 1993). In those cases in which the semen demonstrates nonliquefaction or hyperviscosity, a PCT may be performed. If the results are normal, demonstrating adequate numbers of motile sperm in the cervical mucus, the consistency of the semen may be disregarded. The consistency of the seminal fluid may be of significance if the PCT demonstrates few sperm with good-quality mucus. In these cases, a cross-mucus hostility test or an in vitro cervical mucus–sperm interaction test may be employed. If these tests suggest that either nonliquefaction or hyperviscosity is contributing to the infertility, the best treatment is semen processing and IUI using the male partner’s sperm (artificial insemination with husband’s sperm).Small-volume ejaculates may be produced in patients with obstruction of the ejaculatory ducts, androgen deficiency, retrograde ejaculation, sympathetic denervation, absence of the vas deferens and seminal vesicles, drug therapy, or bladder neck surgery.The significance of high-volume ejaculates remains unclear. Although sperm density may be decreased in high-volume samples, sperm motility remains unchanged (Dickerman et al, 1989). For those cases in which high-volume ejaculates are thought to cause significant low sperm densities, therapeutic insemination of the female partner has been recommended. However, not all investigators agree on this approach. A normal PCT result strongly suggests that a high-volume ejaculate is not a factor in infertility. If high seminal volume is believed to be the etiologic factor, semen processing with concentration of sperm and IUI may be employed.

Sperm Count

A sperm count refers to the concentration of sperm within the seminal plasma. Most methods of sperm count determination utilize counting chambers in which sperm are counted within a grid pattern. Multiple chambers are currently in use. Comparison of methods has revealed significant differences between chambers (Imade et al, 1993; Mahmoud et al, 1997). Because of this, comparison of semen analyses between different laboratories may be difficult. Detailed descriptions of semen analysis techniques are available from several sources (World Health Organization, 1999). Specimens in which no sperm are identified should be centrifuged and the pellet examined for the presence of sperm.

Motility

Motility is the percentage of sperm that demonstrate flagellar motion. The evaluation is ideally performed within 1 to 2 hours of ejaculation and the specimen kept at room or body temperature to avoid a decrease in sperm motility . An assessment of the quality of forward movement of the sperm should be noted. One commonly used method rates the sperm movement on a 5-point scale. A rating of 0 signifies no motility; 1 denotes sluggish or nonprogressive movement; 2 refers to sperm moving with a slow, meandering forward progression; 3 signifies sperm moving in a reasonably straight line with moderate speed; and 4 indicates sperm moving in a straight line with high speed (Amelar et al, 1973). The most common category of sperm movement is reported. An alternate system places the sperm into four categories: A signifies rapid progressive motility; B, slow or sluggish progressive motility; C, nonprogressive motility; and D, no motility. In this system, the percentage of sperm falling into each category is reported (World Health Organization, 1999).It is unnecessary and of no proven prognostic value to determine motility parameters at repeated times after seminal collection. This is a nonphysiologic measurement because sperm leave the semen and enter the cervical mucus within minutes of deposition within the vaginal vault (MacLeod, 1965). The motility may be depressed if the abstinence period has been prolonged. Occasional clumps of agglutinated sperm are of no consequence. However, frequent sperm agglutination is abnormal and suggests the presence of antisperm antibodies. Notation should be made of the presence of other cell types . In the unstained, wet-mount semen specimen, white blood cells and immature germ cells are similar in appearance and are known as round cells. An estimate of the number of these cells per high-power field should be made. Special stains are used to differentiate white blood cells from immature germ cells. (See the section on white blood cell staining.) Cases in which the semen sample demonstrates all nonmotile sperm may be due to ultrastructural defects, in which case the sperm are alive but have defects in the axonemal component of the flagella. Although immotile, these sperm may appear morphologically normal. Alternatively, nonmotile sperm may be dead, in which instance the patient is said to demonstrate necrospermia.

Morphology

The morphologic examination of sperm is a sensitive indicator of the quality of spermatogenesis and of fertility (Talbot and Chacon, 1981; Kruger et al, 1988). Whereas some gross morphologic abnormalities can be identified with bright-field or phase-contrast microscopy of unstained semen, these are very insensitive determinations. Proper morphologic examination involves the use of stained semen specimens . There is no consensus among laboratories as to the classification system of sperm morphology with multiple systems currently in use. Observation of sperm recovered from postcoital cervical mucus or from the surface of zona pellucida has been used to define the appearance of normal sperm (Fredricsson et al, 1977; Mortimer et al, 1982; Menkveld et al, 1990; Liu and Baker, 1992). Older systems have classified the sperm into one of several categories such as normal (oval head), amorphous (irregular head), tapered head, large- and small-headed, and immature ( Fig. 43–1) (MacLeod, 1965). These systems would classify borderline forms as normal and define normal specimens as containing 60% or more normal forms with less than 3% immature forms . Most laboratories currently use more rigid criteria for the definition of a normal sperm ( Fig. 43–2). Borderline forms are considered abnormal (Katz et al, 1986; Kruger et al, 1986).Using these criteria, Kruger and associates (1986)found that in a group of men with sperm densities greater than 20 million/ml and motility greater than 30%, fertilization rates during IVF were 37% for those with less than 14% normal sperm by strict criteria and 91% for those with greater than 14% normal sperm. Furthermore, a breakdown of those patients with less than 14% normal forms demonstrated that those men with less than 4% normal forms had a fertilization rate of 7.6%, whereas those with 4% to 14% normal forms had a fertilization rate of 63.9%. Most, but not all, subsequent studies have reported similar results (Coetzee et al, 1998).Most studies of modern strict criteria of sperm morphology have examined the relationship between morphology and IVF. There have been very limited studies performed on the relationship between strict morphology and conception by intercourse or IUI (Matorras et al, 1995). It remains unclear whether the same normal ranges apply in these situations.

Computer-Aided Semen Analysis

Computer-aided semen analysis (CASA) refers to a semiautomated technique used to individualize and digitalize static and dynamic sperm images using computer-assisted image analysis. Most systems employ video with multiple frames that, when played back, creates moving images. The computerized systems are able to determine parameters not measurable manually.Curvilinear velocity is the average distance per unit time between successive positions of an individual sperm. Straight-line velocity is the speed of a sperm in a forward direction. This is a measure of forward progression and can be correlated with manual methods of forward progression measurement (Yeung et al, 1997). Linearity is determined by dividing straight-line velocity by curvilinear velocity. Additional measurements include lateral head displacement, flagellar beat frequency, and circular movement analysis. Hyperactivation is a state of motility that sperm attain after capacitation in which large-amplitude movements of the head and tail of the sperm are coupled with a slow or nonprogressive motility (Yanagimachi, 1970).Differences in many of these parameters have been found between fertile and infertile men. The advantages of CASA include the ability to get quantitative objective data and the potential for standardization of semen analysis procedures. However, the technique remains without standardization and the results can be affected by many factors. Currently, CASA has not been documented to give a more accurate prognosis or to affect treatment as compared with a manual semen analysis (Davis and Katz, 1993; Krause, 1995).

Additional Semen Parameters

The pH of normal semen is 7.2 or more (World Health Organization, 1999). Semen pH is due to a balance between acidic prostatic secretions and alkaline seminal vesicle secretions. Low ejaculate volume with normal pH may be normal for some patients but may also indicate incomplete collection or retrograde ejaculation, whereas low ejaculate volume and/or acidic pH suggest ejaculatory duct pathology or absent seminal vesicles.

The seminal vesicles produce fructose in an androgen-dependent process. Normal semen fructose concentrations range from 120 to 450 mg/dl. Inflammation of the seminal vesicles, androgen deficiency, partial obstruction of the ejaculatory ducts, or incomplete ejaculation may result in fructose concentrations below 120 mg/dl. In cases of absent seminal vesicles, fructose is usually absent from the semen. Patients with obstructed seminal vesicles or congenital absence of the seminal vesicles, which is usually associated with CBAVD, demonstrate acidic, fructose-negative semen that does not coagulate. In addition, their ejaculate volume is low (<1.0 ml). Transrectal ultrasonography is currently more commonly used to diagnose absence or obstruction of the seminal vesicles and ejaculatory ducts than fructose determinations.Measurements of many other seminal plasma components such as citrate, carnitine, α-glucosidase, fructose, granulocyte elastase, zinc, prostate-specific antigen, glucose, pepsinogen C, insulin-like growth factor, binding protein-3, and prostaglandin D synthase have been performed. Although levels of certain compounds may correlate with spermatogenic activity, these determinations are currently not clinically useful (Diamandis et al, 1999; Chia et al, 2000; Zopfgen et al, 2000).

Interpretation of the Semen Analysis

To properly interpret a semen analysis, the clinician must understand the difference between average semen parameters of a population of normal men and minimal levels of adequacy for normal fertility . Studies of populations of fertile or presumably fertile men report median sperm densities of 70 to 100 million/ml (Irvine et al, 1996; Saidi et al, 1999; Jensen et al, 2000). Considerable controversy has resulted from the publication of a meta-analysis of past studies on sperm counts, which reported a decrease in sperm counts since about 1950 (Carlsen et al, 1992). Further evaluation of the data has suggested that these differences may be due to geographic differences in the populations studied (Fisch and Goluboff, 1996). Analyses of U.S. studies have found no evidence of a decrease in sperm densities over time. Additional studies of European populations have found evidence supporting (Auger et al, 1995; Irvine et al, 1996; Bonde et al, 1998) and refuting (Gyllenborg et al, 1999) a drop in sperm counts over time. Because of the multitude of possible confounding variables in 50-year-old studies, further prospective studies will be required to determine whether sperm counts are truly declining worldwide.The normal range of most diagnostic tests in medicine is determined by using the mean ± 2 standard deviations (SD). Although this may be used to determine “normal” from “abnormal,” this does not separate fertile from infertile men. When semen parameters from populations of fertile and infertile men are compared, there is almost a complete overlap between the two groups. This occurs because fertility is dependent on more than just one semen parameter and on factors affecting both partners.Although a count of 70 million/ml is considered normal, a patient with a count of 70 million/ml but absent motility will be infertile. Studies of couples attempting to conceive have found decreasing probabilities of conception with decreasing sperm counts (Smith et al, 1977; Schoysman and Gerris, 1983; Bonde et al, 1999). Smith and colleagues (1977) reported a 44% pregnancy rate in couples in whom the male had counts between 12.5 and 25 million/ml as compared with a 25% pregnancy rate in those in which the male partner had counts less than 12.5 million/ml. Others have found sperm motility and/or morphology to be equally if not more important than sperm count (Mayaux et al, 1985; Bostofte et al, 1990). In addition, conception is dependent on the fertility status of both partners. Although azoospermic patients are sterile, the majority of infertile men have some motile sperm in their semen and therefore should be considered subfertile rather then sterile.The reference ranges used to interpret the semen analysis are more accurately defined as minimum levels of adequacy. The finding of parameters below these levels is suggestive of infertility, and the finding of parameters above these levels is suggestive of fertility. It is important to keep in mind that there are clearly fertile patients with semen parameters below these levels and infertile patients with parameters above these levels. The World Health Organization (1999) defines the following reference values:

•  Volume: 2.0 ml or more
• pH: 7.2 or more
•  Sperm concentration: 20 × 106  or more sperm/ml
•  Total sperm number: 40 × 106  or more spermatozoa per ejaculate
•  Motility: 50% or more with grade A + B motility or 25% or more with grade A motility
•  Morphology: 15% or more by strict criteria
•  Viability: 75% or more of sperm viable
•  White blood cells: Less than 1 million/ml

Levels that may constitute requirements for natural conception do not apply to the ARTs, in which fertilization may occur with suboptimal semen specimens. Similarly, with the advent of the ability to inject individual sperm into individual ova, it has become clear that the bulk of semen parameters do not predict the fertility of individual spermatozoa.

Hormonal Evaluation

The purpose of the hormonal evaluation of an infertile male patient is to identify endocrinologic disorders that adversely affect male reproduction and to gain prognostic information. Although male reproductive function is critically dependent on endocrinologic control, less than 3% of infertile men have a primary hormonal etiology (Sigman and Jarow, 1997). The most common hormonal abnormality detected on routine testing of infertile men is an elevated serum FSH. In the presence of normal spermatogenesis, FSH secretion is regulated by negative feedback inhibition by the hormone inhibin, which is produced by the Sertoli cells. FSH is often but not always elevated in patients with abnormal spermatogenesis (Turek et al, 1995). Therefore, an elevated serum FSH level is indicative of a significant problem with spermatogenesis whereas a normal serum FSH level does not guarantee intact spermatogenesis. Patients with complete testicular failure have inadequate Leydig and Sertoli cell function that results in elevated gonadotropin levels associated with normal or low testosterone levels ( Table 43–3). Patients with either hypothalamic or pituitary dysfunction have both low serum gonadotropin and testosterone levels as well as absent spermatogenesis (hypogonadotropic hypogonadism).Gonadotropin-releasing hormone (GnRH) is secreted in a pulsatile fashion; and, as a result, gonadotropin hormones are secreted episodically, particularly LH. Some clinicians believe that a proper hormonal assessment requires routinely measuring pooled samples consisting of individual samples taken at 15-minute intervals. Despite the inaccuracies of single determinations, it is rare for a patient’s clinical endocrine status to be inaccurately determined by a single measurement. We recommend a pooled blood sample only when the results of one hormonal determination do not fit the clinical situation (Bain et al, 1988).Throughout early childhood, gonadotropin and testosterone levels remain low. LH and FSH levels begin increasing from 6 to 8 years of age (Conte et al, 1975). Testosterone levels begin increasing at 10 to 12 years of age (Rifkind et al, 1967). During the reproductive years, gonadotropin and testosterone levels remain relatively constant. Later in life, testosterone levels, particularly free testosterone levels, decrease and gonadotropin concentrations rise (Tenover et al, 1987).There is no agreement as to what should constitute the initial endocrine evaluation of infertile men. We recommend that all men with an indication in the history or physical examination or a sperm density less than 10 million/ml have serum FSH and testosterone levels measured, because endocrine abnormalities are rarely present when the sperm concentration is greater than 10 million/ml (Sigman et al, 1997). If these preliminary screening tests are abnormal, further testing with repeat testosterone, prolactin, and LH is indicated. However, some experts believe that all infertile men should undergo endocrine testing and others recommend a more comprehensive panel of tests, including serum LH, prolactin, and thyroid function tests on preliminary screening.If an isolated serum testosterone level is low or borderline and the LH level is not elevated, we recommend morning testosterone and free testosterone tests, because morning levels of testosterone are higher and free testosterone levels are useful in equivocal cases. If the testosterone level remains low, a serum prolactin and pituitary MRI should be obtained (see later). Impaired visual fields or severe headaches suggest the presence of a CNS tumor. Prolactin determination should be performed in these patients. However, not all pituitary tumors are functional and serum prolactin concentration may be normal. Most men with prolactin-secreting tumors have macroadenomas (tumors > 1 cm). Prolactin levels in these patients are typically higher than 50 ng/ml, and both gonadotropin and serum testosterone levels are depressed. Mild prolactin elevations of prolactin (<50 ng/ml) are more frequently discovered in infertile patients, and their clinical significance is questionable. Evaluation of the CNS often fails to identify a tumor in these patients, who often have normal gonadotropin and testosterone levels. Potential causes for mild prolactin elevation includes stress, renal failure, medications, chest wall irritation, and thyroid dysfunction. We do not recommend treatment of isolated mild hyperprolactinemia, because, in our experience, this has not resulted in improved spermatogenesis. However, these patients should undergo an evaluation to rule out a pituitary tumor.

The GnRH stimulation tests may be abnormal in men with suboptimal semen quality, demonstrating an exaggerated pituitary response. However, an abnormal GnRH test does not alter treatment. Therefore, we do not advocate routine GnRH stimulation testing. Likewise, the human chorionic gonadotropin (hCG) stimulation test used to assess Leydig cell function rarely provides diagnostic information beyond routine hormonal tests and is not recommended for routine clinical practice.
Male infertility secondary to congenital adrenal hyperplasia (CAH) is very rare. These patients may have a history of precocious puberty, a family history of CAH, short stature due to premature closure of the epiphyseal plates, and testicular enlargement that may be indicative of adrenal rest tumors of the testis. Measurement of the plasma 17-hydroxyprogesterone reveals elevated levels in these patients. The infertility of male patients with CAH is caused by feedback inhibition of gonadotropin secretion by excessive adrenal androgens that results in suppression of testicular function. Interestingly, many male patients with CAH are fertile (Urban et al, 1978). Partial, late-onset CAH has been found in some cases of male infertility; however, this is uncommon (Augarten et al, 1991). We do not advocate routine screening for CAH.Estrogen excess may be endogenous or exogenous. Measurement of estradiol in men is complicated by the fact that the assay is not very reliable at the low concentrations found in normal men. Testosterone-to-estradiol ratios have been proposed as a method to detect estrogen excess, but normal values for this have not been well established (Pavlovich et al, 2001). Patients with estrogen excess often have bilateral gynecomastia, erectile dysfunction, and atrophic testes. One of the most common causes of estrogen excess is morbid obesity because fat cells contain the enzyme aromatase that converts testosterone to estradiol (Schneider et al, 1979). Estradiol normally stimulates hepatic production of sex steroid hormone–binding globulin (SHBG), which would lower the amount of bioavailable testosterone. Yet many patients with morbid obesity also have hepatic dysfunction and their SHBG levels may be depressed (Glass et al, 1977). Normal levels of plasma FSH, LH, and testosterone are usually found in patients with mildly elevated serum estradiol levels. Thyroid function studies do not need to be determined, because these are generally normal unless there is clinical evidence of thyroid abnormalities.

DIAGNOSTIC ALGORITHMS BASED ON THE INITIAL EVALUATION

Based on the initial history, physical examination, and laboratory studies, a differential diagnosis may be developed ( Table 43–4). Furthermore, more specific testing allows the physician to place the patient into an etiologic category. The frequency of semen abnormalities and etiologic categories in a group of our patients are listed in Tables 43–5 and 43–6.

Absent or Low-Volume Ejaculate

Absent ejaculation may be caused by either retrograde ejaculation or failure of emission (no expulsion of semen through the vas deferens into the posterior urethra) ( Fig. 43–3). The complete absence of seminal fluid is called aspermia, and this disorder should be differentiated from azoospermia (absence of sperm in the seminal fluid). The most common cause of ejaculatory failure is spinal cord injury. Other neurologic disorders such as diabetes mellitus and multiple sclerosis are frequent causes. Medications no longer used for the treatment of hypertension (ganglionic blockers) caused retrograde ejaculation. Retroperitoneal surgery may injure the sympathetic ganglia that control ejaculation, but current methods of nerve-sparing retroperitoneal lymph node dissection avoid this complication in the majority of patients (Foster et al, 1993; Klein, 2000). Another cause of aspermia is psychologic disturbances associated with an inability to obtain orgasm. Low ejaculate volume in the nonazoospermic patient is most often caused by collection problems, which warrants repeat collection. The other main cause is partial retrograde ejaculation, which may be caused by neurologic disorders, medications, or bladder neck surgery or may be idiopathic.Because the majority of seminal fluid is contributed by the seminal vesicles, in the absence of retrograde ejaculation, a low-volume ejaculate suggests the lack of seminal vesicle contribution. Partial or complete ejaculatory duct obstruction will also cause a low-volume ejaculate. Likewise, vasal agenesis, which is often but not always associated with seminal vesicle agenesis, will present with low ejaculate volume azoospermia. Finally, the seminal vesicles and prostate are under androgen control for the production of their secretions. Androgen deficiency will therefore result in reduction of seminal volume. However, all of these causes also produce azoospermia and are discussed in the next section. Some men produce low-volume ejaculates when masturbating into a container but produce normal volumes when ejaculation is accomplished by intercourse. Collection of a specimen with intercourse using a seminal collection condom will identify these cases. Finally, incomplete specimen collection should be ruled out as well as a decreased abstinence period.All cases of absent ejaculation and low-volume ejaculation (<1.5 ml) should be evaluated with a repeat semen analysis and postejaculate urine specimen. The postejaculate urine specimen is evaluated by centrifuging the urine for 10 minutes at 300 g or more. The pellet should then be inspected and interpreted in relationship to the patient’s clinical findings. In patients with absent ejaculation, the finding of greater than 10 to 15 sperm per high-power field indicates retrograde ejaculation. In patients with low-volume ejaculates, the finding of more sperm in the urine than in the antegrade specimen suggests a significant component of retrograde ejaculation. In patients with azoospermia, the finding of any sperm in the postejaculate urine rules out complete bilateral ductal obstruction. In the absence of sperm in the postejaculate urine, ejaculatory duct obstruction should be suspected.

Whereas, traditionally, fructose determination was used to evaluate the presence of seminal vesicle contributions to the ejaculate, transrectal ultrasonography (TRUS) is most commonly employed at the present time (Belker and Steinbock, 1990). Patients with a normal TRUS and low ejaculate volume azoospermia are evaluated like any other patient with azoospermia (described later).Testicular biopsy should be performed if the serum FSH value is normal; and if spermatogenesis is intact, then ductal obstruction is present. To identify the location of the obstruction, a scrotal exploration with vasogram and sampling of vasal fluid is performed at the time of anticipated reconstructive surgery. In low-volume oligospermic patients, there is generally very little reason to perform a testis biopsy, because spermatogenesis must be present. In some of these cases, evidence of ejaculatory duct obstruction may be suggested by the TRUS but partial ejaculatory duct obstruction is an investigative diagnosis at this time. Cases of true partial ejaculatory duct obstruction are extremely rare.

Azoospermia

Azoospermia may be due to inadequate hormonal stimulation (hypogonadotropic hypogonadism), spermatogenic abnormalities, or obstruction. The evaluation of the azoospermic patient should be geared toward determining whether azoospermia is caused by a lack of spermatogenesis or by a ductal obstruction ( Fig. 43–4). The initial step should be to centrifuge the semen specimen.

The presence of any sperm in the pellet rules out bilateral ductal obstruction, and the patient should be evaluated for oligospermia. Although numerous findings on history and physical examination may be predictive of the cause of azoospermia, the main features used to determine the etiology of normal ejaculate volume azoospermia include the presence of the vasa deferentia, testicular size, and serum FSH.

The first step is to determine whether the vasa are present, because CBAVD is a common cause of obstructive azoospermia. CBAVD is a clinical diagnosis based on physical examination and is caused by an abnormality in the CFTR gene (Anguiano et al, 1992; Oates and Amos, 1994). Further radiologic imaging is not routinely necessary, although a small percentage of these patients have upper tract abnormalities and an abdominal ultrasound may be obtained (Schlegel et al, 1996; de la Taille et al, 1998). The vast majority of these patients have normal spermatogenesis such that screening with a serum FSH test alone in the presence of normal testicular volume is sufficient before management. A testicular biopsy may be performed if the history, physical examination, or laboratory studies suggest an abnormality of spermatogenesis.Patients with small testicular volume have either primary or secondary testicular failure. Serum hormone testing including testosterone, LH, FSH, and prolactin is performed to establish this diagnosis as well as to identify both functioning and nonfunctioning pituitary tumors. In patients with small testes and FSH concentrations greater than two to three times normal, severe germ cell failure is present and the ultimate outlook is poor. A testis biopsy should be performed in these patients only if testicular sperm retrieval combined with IVF is being considered. Patients with azoospermia from testicular failure should be offered genetic testing to rule out chromosomal abnormalities such as Klinefelter’s syndrome and microdeletions on the Y chromosome. Patients with secondary testicular failure may be treated with hormone therapy, whereas primary testicular failure is usually irreversible.Finally, patients with vasa present, normal testicular volume, and normal serum FSH levels require testicular biopsy to differentiate between spermatogenic abnormalities and ductal obstruction (Jarow et al, 1989). Scrotal exploration with vasography should be performed at the time of reconstructive surgery and not at the time of testicular biopsy. Patients with a normal-sized testis on one side and an atrophic or absent testis on the contralateral side should undergo testis biopsy even if the FSH value is mildly elevated, because they may have normal spermatogenesis in the larger testis. Patients with unilateral ductal obstruction typically have a normal sperm count and fertility potential. However, this may not be the case if antisperm antibodies have developed because of the obstruction. Occasionally, patients with unilateral obstruction may present with oligospermia or azoospermia owing to an abnormal unobstructed contralateral testis (Matsuda et al, 1992).

Oligospermia

Oligospermia refers to sperm densities of less than 20 million/ml. Isolated oligospermia with normal motility and morphology parameters is uncommon. In cases with sperm counts of less than 5 to 10 million/ml, hormone studies including at least testosterone and FSH should be performed. If these levels are abnormal, a complete endocrine evaluation should be obtained as outlined in the hormonal evaluation section. However, further endocrinologic evaluation is not necessary in oligospermic patients with an isolated elevation of serum FSH, because this indicates abnormal spermatogenesis and is not a true endocrine abnormality. Endocrine abnormalities should be treated appropriately as described in this chapter. Oligospermia as an isolated abnormality is extremely uncommon and most often is caused by androgen deficiency or is idiopathic. Varicoceles are the most common identifiable etiology for oligospermia, but there are frequently abnormalities in other semen parameters as well. Evaluation and treatment of these disorders are discussed in the later section on multiple defects. Treatment options for patients with idiopathic oligospermia include empirical medical therapy and ART.

Asthenospermia

Defects in sperm movement (asthenospermia) refer to low levels of motility or forward progression or both. Spermatozoal structural defects, prolonged abstinence periods, genital tract infection, antisperm antibodies, partial ductal obstruction, varicoceles, and idiopathic causes may be responsible for these cases. (See algorithm in Fig. 43–5.)The finding of asthenospermia or significant sperm agglutination raises the possibility of immunologic infertility. An antisperm antibody assay, preferably a direct assay, should be performed. Patients with antisperm antibodies are most commonly directed toward ARTs. Although success with immunosuppressive regimens has been reported, their overall efficacy is low and there is a risk of serious side effects such as aseptic hip necrosis. Hormonal studies are not indicated for isolated asthenospermia. Infections should be suspected if increased numbers of round cells have been reported in the semen analysis. In these instances, a study to differentiate immature germ cells from white blood cells, such as the Endtz test or immunohistochemical staining, may be considered. Semen cultures or urethral swabs to detect the presence of Mycoplasma and Chlamydia may be performed to further evaluate true pyospermia. In addition, a urinalysis should be performed to rule out the presence of a urinary tract infection. Semen cultures may be performed but are often contaminated by distal urethral organisms (Kim and Goldstein, 1999).A varicocele is the most common surgically correctable abnormality found in infertile men and may be responsible for sperm motility defects as well as defects in sperm count and shape. Partial ejaculatory duct obstruction may be suspected in asthenospermic patients, particularly in the presence of low ejaculate volume and low viability. TRUS may be performed in these instances; and when findings are abnormal, further evaluation for ejaculatory duct obstruction should be performed.Complete absence of sperm motility or cases with motilities less than 5% should be evaluated by a sperm viability assay. The finding of a high fraction of viable sperm in the presence of low or absent sperm motility suggests an ultrastructural abnormality, such as that found in the immotile cilia syndrome and Kartagener’s syndrome (immotile cilia syndrome associated with situs inversus). Patients with classic immotile cilia syndrome typically have a history of chronic respiratory infections. This is because of a lack of dynein arms in the axoneme of the cilia of the respiratory tract and in the flagella of the spermatozoa. Situs inversus is present in approximately 50% of these patients (see section on structural abnormalities of spermatozoa). Electron microscopic evaluation of spermatozoa identifies these cases. Occasionally, excessively prolonged abstinence periods may result in severely depressed motility. Finally, toxic contaminants in the collection container or exposure of the specimen to hot or cold environments may also account for low motility.

Teratospermia

Defects in morphology are termed teratospermia and are found more commonly with the increased use of rigid criteria for the evaluation of sperm morphology. Teratospermia is often associated with both oligospermia and asthenospermia. Temporary insults to spermatogenesis and varicoceles are potential causes. Rarely, patients may have round-headed sperm owing to absence of the acrosome.

Multiple Defects in Seminal Parameters

Combined defects in sperm density, motility, and morphology are known as oligoasthenoteratospermia and are most frequently caused by a varicocele effect. Varicoceles are diagnosed by physical examination and are graded as small, medium, and large. Radiologic imaging to detect a varicocele in patients without clinical evidence of one is not routinely indicated. However, either scrotal ultrasonography or venography may be used in patients who are difficult to examine or to detect the presence of a persistent varicocele after treatment.Other causes of multiple sperm defects include cryptorchidism, temporary insults to spermatogenesis such as heat, drugs, or environmental toxins, or idiopathic causes. A heat effect may be either environmental or endogenous, such as caused by a fever. If a temporary insult is suspected, several semen analyses over one to two spermatogenic cycles (3 to 6 months) should be obtained once the inciting agent is removed.As mentioned previously, partial ejaculatory duct obstruction may be a cause of low seminal volume associated with multiple defects in semen parameters (Meacham et al, 1993). TRUS and seminal vesicle aspiration have been advocated to diagnose this disorder (Jarow, 1994). However, this diagnosis is very difficult to document and many patients who are thought to have partial obstruction likely have idiopathic infertility. Partial ejaculatory duct obstruction should be considered an investigational diagnosis at this time.

Normal Semen Parameters

Normal semen analyses in infertile couples suggest the possibility of a female factor as well as immunologic infertility or incorrect coital habits. A PCT should be obtained in these instances. If no sperm are identified, the couple must be questioned about their coital technique. The presence of a shaking motion of sperm in the cervical mucus is suggestive of antisperm antibodies. If the evaluation of both partners is normal, the couple has unexplained infertility. In these instances, both partners should be evaluated for the presence of antisperm antibodies. A direct assay is preferred in the male, and an indirect one is performed in the female. The female should also be carefully evaluated to rule out significant female factors. Rarely, men with normal semen parameters have a functional defect that prevents their sperm from fertilizing eggs in vivo and in vitro. Sperm function testing by either the sperm penetration assay or the acrosome reaction test may be performed in the male partner of a couple with unexplained infertility. Abnormal sperm function testing results should instigate a reevaluation of the male in a search for correctable abnormalities. If no abnormalities are detected, the IVF with ICSI should be considered.

ADDITIONAL TESTING

Based on the initial evaluation and the differential diagnosis suggested by this evaluation, additional more specific testing may be indicated, as outlined in the algorithms depicted earlier. The purpose of these additional tests is to rule in or out specific causes for male infertility. It is important to keep in mind that there are specific indications for each of these tests and that they do not need to be performed on a routine basis.

Antisperm Antibodies

As spermatogenesis begins in the pubertal male, tight junctions develop between Sertoli cells in the seminiferous tubules forming the basis of the blood-testis barrier. This barrier prevents the immune system from coming in contact with sperm surface antigens that are present on postmeiotic germ cells. T-suppressor cells and additional immunosuppressive factors such as prostaglandin E2 in the seminal plasma may also be involved in suppressing the immune response to sperm antigens (el Demiry et al, 1988; Quayle et al, 1989).Risk factors for the development of antisperm antibodies include conditions that may disrupt the blood-testis barrier. Approximately 60% of men develop antisperm antibodies after vasectomy (Ansbacher, 1971; Fuchs and Alexander, 1983), whereas approximately one third of patients with CBAVD are found to have antisperm antibodies present on the sperm at the time of retrieval from the epididymis (Patrizio et al, 1992). Other causes that have been associated with the presence of antisperm antibodies include acute epididymitis, cryptorchidism, and genital trauma (Iqbal et al, 1989; Heidenreich et al, 1994; Urry et al, 1994).

Although there is some evidence suggesting testicular torsion is a risk factor for the presence of antisperm antibodies (Sinisi et al, 1993a), most studies have not demonstrated this association (Henderson et al, 1985; Puri et al, 1985; Hagen et al, 1992). Similarly, there is evidence both for (Gilbert et al, 1989; Knudson et al, 1994) and against (Oshinsky et al, 1993) an association between varicoceles and antisperm antibodies. Of interest, in one report, a high incidence of serum antisperm antibodies against epididymal sperm was noted, whereas there was a lower incidence of antisperm antibodies against ejaculated sperm in patients with varicoceles. This suggests that epididymal sperm antigens may play a role in some cases of varicocele-associated immunologic infertility (Fichorova et al, 1995). Correlations have also been noted between the presence of antisperm antibodies and homosexual rectal intercourse (Witkin et al, 1983; Mulhall et al, 1990), sexually transmitted diseases (particularly pastChlamydial infections) (Close et al, 1987; Hargreave et al, 1984; Witkin et al, 1995), testis cancer (Foster et al, 1991), and a history of testicular biopsy (Hjort et al, 1974).

Abnormal PCTs, particularly when immotile sperm with a shaking motion are noticed, are highly suggestive of the presence of antisperm antibodies (Matson et al, 1988; Menge et al, 1989; Pretorius and Franken, 1989; Kremer and Jager, 1992). Couples with unexplained infertility as well as cases with impaired sperm motility or sperm agglutination have also been reported to have a higher incidence of antisperm antibodies (Menge et al, 1989; Cimino et al, 1993; Omu et al, 1999). Immunoglobulins may enter the genital tract through the seminiferous tubules, epididymis, or prostate. Immunoglobulin A (IgA) and IgG may both passively diffuse into the genital tract; however, IgA is also actively secreted. Because immunoglobulins may be secreted locally in the genital tract, it is not surprising that there may be antisperm antibodies present on the sperm surface that are not found in the serum.

There are many assays to test for the presence of antisperm antibodies. Direct assays detect the presence of antisperm antibodies on the patient’s sperm. Indirect assays measure antisperm antibodies in the patient’s serum and generally require antisperm antibody–negative donor sperm. Because it is the sperm, not serum, that reach the female reproductive tract, direct assays have an advantage of detecting only sperm-bound immunoglobulins. The presence of antisperm antibodies in the serum is not always associated with the presence of these antibodies on sperm (Hellstrom et al, 1988). In addition, IgM class antibodies that may be present in serum do not usually make it to the semen.

In general, the higher the titer of antibodies in the serum, the more likely there will be antibodies in semen. Most investigators believe that only antibodies present on the spermatozoal surface are clinically significant. Thus, most recent investigations have been aimed at direct assays determining the presence of sperm-bound antibodies instead of the indirect detection of serum antisperm antibodies ( Table 43–7). The immunobead assay and the mixed agglutination reaction are commonly used for the detection of antisperm antibodies. These assays utilize bead or red blood cells that will bind to antibodies bound to the sperm surface. Scoring is based on the percentage of motile sperm with bead or red blood cell binding (Ayvaliotis et al, 1985; Clarke et al, 1985a). Most laboratories consider a sample positive for the presence of antisperm antibodies if more than 20% to 50% of the sperm demonstrate binding. An indirect assay may be performed by using patient’s serum with donor sperm. Currently, clinically used antisperm antibody assays do not differentiate between antibodies directed against different sperm surface antigens. It is likely that the variable effects of antisperm antibodies may, in part, depend on which antigen the antibody is directed against. It is hoped that future assays may allow for differentiation between clinically significant and insignificant antisperm antibodies.

Antisperm antibodies can affect sperm function at several different levels. Cervical mucus penetration may be impaired in sperm coated with antibodies. There appears to be some interaction between the cervical mucus and the Fc region of IgA antibodies (Jager et al, 1980). Effects of antibodies have included inhibition of sperm capacitation (Benoff et al, 1993), premature induction of the acrosome reaction, or impairment of zona binding or fertilization of the ova (Clarke et al, 1985b; de Almeida et al, 1989; Mahony et al, 1991). There is evidence that IgA antibodies directed against sperm tails significantly impair sperm transport through cervical mucus. In contrast, IgG or IgA antibody directed against sperm heads may not impair transport but may interfere with fertilization. However, this impairment appears to be less complete than the degree to which anti-tail IgA effects sperm transport.

Antisperm antibodies are detected in approximately 10% of males presenting with infertility as opposed to 2% or less of fertile men (Clarke et al, 1985b; Sinisi et al, 1993b). The greater the amount of antisperm antibodies, the greater the effect on fertility. Similarly, low concentrations of antibodies seem to have little effect on fertility (Rumke et al, 1974; Ayvaliotis et al, 1985). Although assays such as the immunobead assay determine the percentage of sperm with antibody bound to their surface, they do not give a measurement of the amount of antibody bound to the sperm. Indirect assays yield this information, and, therefore, some have suggested direct assays be used as a screening test (because of the their high specificity) followed by indirect assays to determine serum titers (Hjort, 1999).

Fertility may also be impaired in the presence of antisperm antibodies in the female. Of necessity, indirect assays must be used on females and therefore are subject to the same problems as indirect assays in the male. Although serum is easier to work with, antisperm antibodies in the cervical mucus are more clinically significant; however, this substance is technically difficult to work with.
We believe that patients with the previously mentioned risk factors, those demonstrating impaired sperm motility, sperm agglutination, abnormal PCT findings, and couples with unexplained infertility, should be tested for the presence of antisperm antibodies. Because the presence of serum antisperm antibodies does not affect the decision to proceed with vasectomy reversals, we do not recommend preoperative testing in these patients. Testing may be performed on patients with patent anastomoses after reversals who are not able to impregnate their partners within 1 year.

Leukocyte Staining

Under wet-mount microscopy, immature germ cells and leukocytes appear similar and are known as round cells. The presence of greater than 1 million white blood cells/ml is considered abnormal and raises the possibility of a genital tract infection or inflammation. Yet, the concentration of white blood cells in the fertile and infertile populations overlap considerably. Both infection and infertility have been associated with pyospermia (Caldamone et al, 1980; Berger et al, 1982; Maruyama et al, 1985).

The concentration of immature germ cells is proportional to the concentration of sperm, although many men with poor semen quality have a higher percentage of immature cells than fertile men. However, the patient-to-patient variation is considerable and is of unclear significance (Wolff et al, 1990). Most studies suggest detrimental effects of leukocytes on sperm function and semen parameters (Yanushpolsky et al, 1996). However, many patients with pyospermia do not have genital tract infections. Approximately one third of the cases demonstrating increased numbers of round cells will be found to have true pyospermia, with the remainder of cases having increased numbers of immature germ cells (Sigman and Lopes, 1993). Although infertile couples tend to have greater concentrations of white blood cells than fertile populations (Wolff and Anderson, 1988), not all studies of patients with increased numbers of leukocytes in the semen report decreased fertility rates (Tomlinson et al, 1993).

Morphologic differentiation of round cells may be made using traditional staining techniques, such as the Papanicolaou stain. However, these methods necessitate a highly trained observer. Monoclonal antibodies directed against white blood cell surface antigens have been combined with immunohistochemical techniques to aid in the differentiation of round cells (Homyk et al, 1990). The red-brown staining pattern of leukocytes allows for their easy differentiation from immature germ cells (Wolff et al, 1988). Histologic techniques relying on the presence of peroxidase within the white blood cells underestimate the number of leukocytes within semen because some cells do not contain this enzyme (monocytes). Peroxidase-positive white blood cells stain dark brown with this simple procedure (Endtz, 1974). We recommend some type of white blood cell staining of semen in patients with more than 10 to 15 round cells per high-power field or more than 1 million round cells/ml. If the majority of cells are white blood cells and their concentration is greater than 1 million cells/ml, the patient should be evaluated for a genital tract infection.

The management of pyospermia, in the absence of genital tract infection, has included anti-inflammatory medication, empirical antibiotic therapy, frequent ejaculations, and prostatic massage. However, these therapies lack proven efficacy (Yanushpolsky et al, 1995). Management with ARTs that includes processing to remove the white blood cells should be considered in these cases.

Semen Cultures

Genital tract infections are uncommon causes of male infertility. Many organisms, including aerobic and anaerobic organisms, as well as Mycoplasma, have been cultured from human semen (Swenson et al, 1980; Lewis et al, 1981; Busolo et al, 1984b; Naessens et al, 1986; Upadhyaya et al, 1984). Although studies have found that bacteria may be spermicidal (Paulson and Polakoski, 1977), others have found no consistent effect on fertility (Berger et al, 1982; Makler et al, 1981). In the presence of a culture-positive genitourinary infection, such as cystitis, urethritis, or prostatitis, appropriate treatment should be instituted. However, routine genital tract bacterial culturing is not indicated in the absence of clinical symptoms or documented pyospermia.

Mycoplasmas are aerobic bacteria of the family Mycoplasmataceae, which includes the genera Mycoplasma and Ureaplasma. These bacteria bind to cell membranes and do not stain with Gram stain. Both Mycoplasma hominis and Ureaplasma urealyticum have been associated with nongonococcal urethritis in humans (Bowie et al, 1977). There are conflicting studies on the relationship between the presence ofMycoplasma in the genital tract and semen parameters (Upadhyaya et al, 1984; Soffer et al, 1990). Studies have demonstrated decreased motility and membrane changes in semen samples withUreaplasma, which seems to attach to the head and midpiece of some spermatozoa (Nunez-Calonge et al, 1998). Similarly, some investigators have found evidence of adherence ofMycoplasma to sperm (Lewis et al, 1981; Busolo et al, 1984a; Upadhyaya et al, 1984). Although uncontrolled studies have reported improved pregnancy rates in couples in which the male was treated for these infections (Kundsin et al, 1986), controlled studies have not confirmed these findings (Idriss et al, 1978).
Chlamydia, a common obligate intracellular bacterium, is a frequent cause of epididymitis and nongonococcal urethritis. Cultures of semen, prostatic secretions, and urine have documented the presence of Chlamydia (Thompson and Washington, 1983). A higher prevalence ofChlamydia has been found in asymptomatic patients with unexplained infertility as compared with fertile patients (Greendale et al, 1993). In addition,Chlamydia have been found to bind to and penetrate human spermatozoa.

Our approach is to test patients with clinical evidence of an inflammatory or infectious process for Mycoplasma and Chlamydia. Semen may be cultured for bacterial organisms; however, these frequently yield low concentrations of multiple organisms owing to distal urethral contamination. Antibacterial skin preparation and voiding before ejaculation decreases the incidence of, but does not eliminate, false-positive cultures (Kim et al, 1999). We do not recommend routine cultures in patients without evidence of disease. Urine cultures should also be obtained in those patients with evidence of cystitis or urethritis.

Ultrastructural Evaluation

Electron microscopy is used to evaluate the ultrastructure of spermatozoa. Light microscopy detects gross tail abnormalities, such as bent or coiled tails as well as absence of the acrosome. Electron microscopy is necessary for detection of abnormalities of mitochondria, outer dense fibers, or microtubules. These defects generally result in absent or extremely low motility. In these cases, a sperm viability assay should be performed. This will differentiate dead sperm from live nonmotile sperm. Electron microscopy should be considered only in those patients with low or absent motility and high viability scores. Ultrastructural abnormalities have also been identified in cases with apparently normal sperm morphology by light microscopy but unexplained failure of in vitro fertilization (Carlon et al, 1992). Electron microscopy should be considered for patients with sperm samples with very low motility but reasonably high viability or in occasional cases of unexplained infertility.

Radiologic Evaluation

The main purpose of the radiologic evaluation of the genital system of the infertile male is to identify evidence of either complete or partial ductal obstruction. A patient with complete obstruction of the excurrent ductal system typically has azoospermia, whereas patients with partial obstruction can have a variety of seminal findings, including oligospermia, asthenospermia, and/or early demise of sperm after ejaculation. It is extremely difficult to differentiate partial ductal obstruction from other causes of male infertility, including idiopathic oligospermia. Moreover, the currently available radiologic imaging studies cannot provide a definitive diagnosis for partial obstruction. Thus, the diagnosis and management of patients thought to have partial ductal obstruction is considered investigational.

Vasography

The traditional and most commonly employed radiologic imaging study employed for the evaluation of the vasal and ejaculatory duct patency is vasography. Vasography is indicated to determine the site of obstruction in azoospermic patients who have active spermatogenesis documented by testis biopsy. Vasography is best performed in conjunction with reconstructive surgery because this procedure carries an inherent risk of vasal injury that could complicate future reconstructive surgery if performed separately (Poore et al, 1997). A retrograde method employing endoscopic canalization of the ejaculatory ducts is no longer used because of both technical difficulties and the risk of epididymitis when contrast medium is injected into the epididymis.

Vasography is most commonly performed at the level of the scrotal vas deferens either by puncture or through a transverse vasotomy. The site of obstruction is determined by the combination of injection of an agent distally and microscopic inspection of the intravasal fluid to determine epididymal patency. Injection of plain saline solution or saline solution combined with a colored dye is used initially to document distal patency. However, if the saline solution does not pass easily, injection of dilute nonionic contrast agent or passage of a 2-0 monofilament suture is necessary to determine the site of obstruction.

A normal vasogram is documented when contrast agent is visualized throughout the length of the vas deferens, seminal vesicles, ejaculatory duct, and bladder ( Fig. 43–6A and B). Proximal patency of the epididymis is documented by microscopic (×400) visualization of sperm in the intravasal fluid. This procedure is discussed in detail in Chapter 44, Surgical Management of Male Infertility and Other Scrotal Disorders. Vasography may also be indicated in the severely oligospermic patient when there is reason to suspect a unilateral vasal obstruction (such as from a hernia repair) with an abnormal contralateral testis (Matsuda et al, 1992).
An alternative approach to detect ejaculatory duct obstruction is seminal vesiculography (see Fig. 43–6C and D). However, there is a significant risk of introducing infection into a closed system, particularly if seminal vesicle puncture is performed transrectally rather than transperineally. Therefore, it is best to perform this diagnostic procedure at the time of intended relief of obstruction.

Seminal vesiculography can also be used in patients with suspected obstruction of the inguinal portion of the vas deferens but should not be used to evaluate the scrotal vas deferens, because of the risk of epididymitis (Riedenklau et al, 1995). Vasography is not indicated in the oligospermic patient without evidence of vasal obstruction by history or physical examination and symmetrical testes.

Transrectal Ultrasonography

TRUS allows for the anatomic visualization of the prostate, seminal vesicles, and ampullary portion of the vas deferens ( Fig. 43–7). TRUS is indicated in azoospermic patients suspected of having ejaculatory duct obstruction. The typical seminal findings in patients with complete ejaculatory duct obstruction include low ejaculate volume (<1 ml), acidic pH, absent fructose, and failure to coagulate because of the absence of seminal vesicle secretions. The differential diagnosis for these seminal findings includes both ejaculatory duct obstruction and vasal agenesis associated with seminal vesicle agenesis or hypoplasia. Obstruction of the ejaculatory ducts is suggested by the presence of dilated seminal vesicles. The normal diameter of the seminal vesicles on transverse imaging behind the bladder is up to 1.5 cm (Carter et al, 1989). Hypoplasia or absence of the seminal vesicles is easily diagnosed, but some patients with complete ejaculatory duct obstruction do not have dilated seminal vesicles (Jarow, 1996a). Either vasography or seminal vesicle aspiration may be necessary to establish the diagnosis of ejaculatory duct obstruction in patients with equivocal ultrasonographic findings.
Seminal vesicle aspiration is performed under TRUS using a 30-cm or longer 20-gauge needle. The presence of millions of sperm in the seminal vesicle aspirate of an azoospermic man is diagnostic of ejaculatory duct obstruction (Jarow, 1996b). Moreover, the presence of sperm in these patients obviates the need for testicular biopsy to confirm the presence of active spermatogenesis and rules out the presence of concomitant epididymal obstruction (Silber, 1980).

It has been suggested by some investigators that select infertile patients have partial ejaculatory duct obstruction (Hellerstein et al, 1992; Goluboff et al, 1995; Beiswanger et al, 1998). The clinical findings that are thought to be consistent with partial ejaculatory duct obstruction include normal or low-normal semen volume, reduced motility, and early demise of sperm in vitro in patients with normal hormonal profiles and normal-sized testes (Hellerstein et al, 1992). Although the finding of dilated seminal vesicles greater than 1.5 cm associated with a dilated ejaculatory duct is suggestive of ejaculatory duct obstruction, it is notdiagnostic. In addition, it has been suggested that intraprostatic cysts and hyperechoic lesions within the prostate are associated with ejaculatory duct obstruction. Yet, hyperechoic lesions of the prostate are frequently seen in fertile volunteers and intraprostatic cysts can be an incidental finding (Jarow, 1993; Poore and Jarow, 1995). It has also been suggested that the finding of a large number of motile sperm in a seminal vesicle aspirate is consistent with partial ejaculatory duct obstruction, but this assertion remains unproved (Jarow, 1994). The indications for TRUS in patients with suspected partial ejaculatory duct obstruction as well as the ultrasonographic criteria to diagnose this condition remain quite controversial, and treatment of these patients should be considered investigational at this time (Jarow, 1996a).

Venography

Internal spermatic vein venography is used to both detect and potentially treat varicoceles. Venography is performed using Seldinger technique through either a right femoral venous or a right internal jugular venous approach. The femoral vein approach is generally preferred, but the internal jugular approach is superior if embolization of bilateral varicoceles is being contemplated. Venography should be performed under low pressure and, for the left side, with the catheter tip positioned inside the renal vein lateral to the junction with the internal spermatic vein ( Fig. 43–8).

There are a number of potential false-positive and false-negative results with venography because of technical errors and anatomic variations. The valves of the internal spermatic veins are often located very close to the ostium, and a false-positive result may be obtained if the vein is cannulated or extreme pressures are used during injection of contrast material (Nadel et al, 1984). In addition, there are frequently multiple openings of the internal spermatic veins at their origin. Both of these features may lead to a false-positive diagnosis of a varicocele by venography. Conversely, a false-negative study may occur if the patient is not studied in the reverse Trendelenburg position or if the reflux is the result of collateral veins not visualized during the study (Wishahi, 1991). Although there is considerable controversy regarding the role of venography in the management of varicoceles, most clinicians reserve venography for patients with a suspected recurrence after varicocele repair both to document the recurrence and to embolize the persistent veins.

Scrotal Ultrasonography

The main application of scrotal ultrasonography in male infertility has been for the diagnosis of varicoceles. Varicoceles are normally detected by physical examination, but some patients may be difficult to examine or have equivocal results of the examination. Color duplex scrotal ultrasonography has been applied as a noninvasive alternative to internal spermatic vein venography in an attempt to objectively diagnose varicoceles. Other noninvasive tests for varicoceles include the Doppler stethoscope, thermography, and radionucleotide studies (World Health Organization, 1985).

The initial criteria developed to diagnose a varicocele include the presence of numerous large veins (>3 mm) and reversal of blood flow with Valsalva maneuver (McClure and Hricak, 1986). However, further studies have shown that the accuracy of color duplex ultrasonography diagnosis of varicoceles is only 60% when compared with both physical examination and venography (Eskew et al, 1993). Moreover, there is little evidence that repair of subclinical varicoceles has any positive effect on male fertility (Jarow et al, 1996; Yamamoto et al, 1996). Therefore, there is limited value in using scrotal ultrasonography for the detection of varicoceles in subfertile men.

Imaging studies should not be used to search for varicoceles in men with normal and adequate physical examinations. Color duplex scrotal ultrasonography should be reserved for those patients with an inadequate physical examination because of either obesity or testicular sensitivity. A venous diameter of 3.5 mm or greater should be used as the criterion to diagnose a clinical varicocele in these patients (Eskew et al, 1993; Meacham et al, 1994). The other application of scrotal ultrasonography in the subfertile patient is to rule out the presence of testicular tumors. Subfertility is sometimes a presenting symptom of testicular germ cell neoplasia, and scrotal ultrasonography is the best radiologic imaging modality for this problem (Honig et al, 1994). In addition, Leydig cell tumors are often not palpable and should be suspected in subfertile men with a high serum testosterone and/or estradiol level or gynecomastia (Horstman et al, 1994; Lemack et al, 1995). Scrotal ultrasonography to detect testicular tumors should be restricted to patients with suggestive histories, physical examinations, or hormonal values. It should not be used as a routine examination to screen all infertile men.

Abdominal Ultrasonography

Abdominal ultrasonography is used to assess the kidneys in patients with an absent vas deferens. The vas deferens and ureter share an embryologic origin in the mesonephric duct. Hence, patients with congenital absence of the vas deferens are also at risk for renal agenesis. Ipsilateral renal anomalies are present in up to 80% of men with unilateral absence of the vas deferens, with the most common anomaly being renal agenesis (Donohue and Fauver, 1989). In contrast, the mechanism of CBAVD appears to be through a different mechanism and the risk for renal anomalies is much lower (Schlegel et al, 1996). Therefore, abdominal ultrasonography should be considered to rule out renal agenesis in patients with a nonpalpable vas deferens.

Sperm Function Testing

Sperm–Cervical Mucus Interaction

For conception to occur after intercourse, sperm must travel through the cervical mucus. The PCT assesses this interaction. The examination should be performed just before ovulation, at which point the cervical mucus becomes clear and thin. A drop of cervical mucus is examined under a microscope (Moghissi, 1976). Although the test has been used since the 1940s, there is no agreement as to how the test should be performed, the timing of the test, or the grading system.
A normal test result is usually defined as one in which more than 10 to 20 sperm are identified per high-power field (×400). Progressive motility should be present in the majority of sperm. Most investigators agree that in the face of normal PCT findings, a cervical factor or semen deposition abnormality is not involved in the couple’s infertility. However, abnormal PCT may result from many causes. Inappropriate timing of the PCT is the most common cause for an abnormal result. Other causes include anatomic abnormalities, semen or cervical mucus antisperm antibodies, inappropriately performed intercourse, and abnormal semen.

Because of the lack of standardization, reproducibility, and the increasing popularity of ARTs, the usefulness of the PCT in the evaluation of the infertile couple has come into question (Glatstein et al, 1995). Prospective studies examining the value of the PCT in predicting subsequent fertility have given conflicting results with only some studies supporting its value (Jette and Glass, 1972; Lyon et al, 1982; Collins et al, 1984).

In an attempt to standardize the cervical mucus interaction, in vitro cervical mucus tests have been developed. In the slide test, a drop of sperm is placed adjacent to cervical mucus under a microscope. The ability of the sperm to penetrate the mucus is then examined microscopically. This test is simple; however, it may be difficult to reproduce and is not quantifiable. To quantitate the cervical mucus interaction more easily, cervical mucus may be placed in a capillary tube, which is then placed in contact with the semen specimen. The migration distance, penetration density, and quality of progressive movement can then be determined.

Finally, to localize the etiology of an abnormal PCT to either partner, cross-mucus hostility testing may be performed. In this procedure, the woman’s mucus is placed in contact with donor sperm as well as with the partner’s sperm. Similarly, the partner’s sperm, as well the donor’s sperm, is placed in contact with donor’s mucus (Morgan et al, 1977). This test is limited by the availability of donor sperm and donor mucus. To completely remove the female factor from the assay, bovine cervical mucus has been used for in vitro testing (Gaddum-Rosse et al, 1980; Alexander, 1981; Mangione et al, 1981; Moghissi et al, 1982). Despite the additional information that may be obtained from these assays, they are not commonly used in most couples.

The quality of the semen specimen (particularly motility parameters) correlates with the quality of the cervical mucus penetration tests. However, poor cervical mucus interaction tests have been found in some patients with normal semen parameters (Takemoto et al, 1985; Keel et al, 1987). The PCT test is indicated in cases of hyperviscous semen, unexplained infertility, and low-volume or high-volume semen specimens with normal total sperm counts. Because patients with very poor quality semen invariably have poor PCTs, it is not necessary to perform PCTs in this group of patients. A persistently abnormal PCT in the face of reasonably good semen parameters should lead the physician to question the quality of the cervical mucus.

The quality of the mucus is rated by inspecting its ferning and spinnbarkeit. The gynecologist usually performs this at the time of the PCT. If the mucus quality is reported to be good, then poor timing relative to the time of ovulation is not likely to be involved. If no sperm are seen in good-quality cervical mucus, the couple should be questioned about their coital technique and the physician should be sure that the patient does not have hypospadias, which would lead to a sperm deposition problem. The finding of good-quality mucus and few or nonmotile sperm or immobilized sperm demonstrating a shaking motion should lead to the evaluation of both the male and the female partner for the presence of antisperm antibodies. An in vitro cervical mucus interaction test may be considered after an abnormal PCT to further localize the source of the defect. Currently, many physicians would proceed with IUI rather than perform additional diagnostic tests.

Acrosome Reaction

Fertilization requires sperm to undergo capacitation and the acrosome reaction. Visualization of the human acrosome requires specific staining to differentiate acrosome-reacted sperm from acrosome-intact sperm. Transmission electron microscopy clearly defines the status of the acrosome, but it is an expensive and labor-intensive procedure and is not suitable for routine clinical use. Traditional tissue-staining techniques have been developed to examine the acrosome (Talbot and Chacon, 1981). A recent assay has combined the hyposmotic swelling test with acrosome staining to produce a much less labor-intensive procedure (Ohashi et al, 1995; Glazier et al, 2000). Many other additional techniques have also been used (Talbot and Chacon, 1981; Kallajoki et al, 1986; Lee et al, 1987; Mortimer et al, 1987; Cross and Meizel, 1989).

Sperm must undergo capacitation before the acrosome reaction. Although in vivo capacitation normally occurs over 4 to 6 hours within the female reproductive tract, it takes approximately 3 hours under in vitro capacitation conditions. After capacitation, sperm may be induced to undergo the acrosome reaction by exposing them to acrosome-inducing agents. Acrosome reaction studies may determine the percentage of cells in the semen sample that have spontaneously undergone the acrosome reaction as well as the percentage of cells that may be induced to undergo the acrosome reaction after capacitation and exposure to an inducing agent.

In general, normal semen samples demonstrate a spontaneous acrosome reaction rate of less than 5% and an induced acrosome reaction rate of 15 to 40%. Samples from infertile populations have demonstrated high spontaneous rates of acrosome-reacted sperm and low inducibility of the acrosome reaction (Fenichel et al, 1991). Acrosome reaction assays are not widely available; however, they may be considered in patients with unexplained poor fertilization rates obtained in IVF or in cases of unexplained infertility. If an acrosome reaction defect is identified, IVF with ICSI is indicated. Because normal penetration in a sperm penetration assay (SPA) requires the sperm to undergo the acrosome reaction, an SPA may be performed if an acrosome reaction assay is not available.

Sperm Penetration Assay

Cross-species fertilization is prevented by the zona pellucida. This glycoprotein layer surrounds the ovum of most species. The removal of this layer allows human sperm to fuse with hamster oocytes. For penetration to occur, the sperm must be capacitated in vitro. Scoring is performed by determining the percentage of ova that have been penetrated or by calculating the number of sperm that have penetrated each ovum. Thus, this assay requires sperm to be able to undergo (1) capacitation, (2) the acrosome reaction, (3) fusion with the oolemma, and (4) incorporation into the ooplasm. Because the zona has been removed, this test will not detect abnormalities of sperm-zona interaction.

Samples are commonly believed to be normal if the sperm penetrate between 10% and 30% of ova. Unfortunately, the assay is a bioassay with variable results and is not standardized. Therefore, there are conflicting results and significant controversies as to its interpretation. A modification of the SPA procedure, involving incubation of the sperm in a more potent capacitating media, results in the majority of oocytes being penetrated. Scoring for these assays is based on the number of penetrations per ova. SPA results using this approach have correlated well with pregnancies after intercourse in male factor couples with a positive predictive value of 77.8% and a negative predictive value of 92.3% (Gattuccio et al, 1988). Others have found similarly good correlations between the results of the SPA and fertilization and pregnancy rates by conventional IVF (Smith et al, 1987; Shibahara et al, 1998). For proper interpretation of the SPA, the physician must be familiar with the laboratory that is performing the assay and should be aware of what correlations have been documented between the results of the assay and actual human fertilization.

We believe the SPA should be obtained in men with low numbers of morphologically normal sperm to rule out a fertilization defect. Some clinicians also obtain SPAs in couples with unexplained infertility. Those couples with abnormal SPA results should consider IVF with ICSI as opposed to IUI or conventional IVF (Shibahara et al, 1998).

Other Sperm Function Tests

Hemizona Assay

In the hemizona assay, the human zona pellucida is microscopically divided in half. Each half is then incubated with either patient sperm or sperm from a fertile donor. A hemizona index is then calculated by counting the number of sperm bound to each zona half and dividing the number of sperm bound from the patient by the number of sperm bound from the donor. Most male patients who do not fertilize human ova in vitro demonstrate a hemizona index of less than 0.60 (Burkman et al, 1988). Because this assay requires a source of human ova and significant micromanipulation skills, it has not gained widespread use. Both zona pellucida derived from cadavers and from failed IVF cycles have been used to increase the availability of zona for this assay (Franken, 1998; Henkel et al, 1999). This test will determine whether a zona-sperm interaction defect is present in cases in which IVF has not resulted in fertilization in the presence of a normal SPA. Patients demonstrating defects in the hemizona assay should be referred for IVF with ICSI. However, with the availability of ICSI, this test has become unnecessary and is not often performed.

Sperm Viability Assays

Nonmotile sperm may be viable but lack the ability to move or may be dead. The finding of a predominance of viable nonmotile sperm suggests the presence of ultrastructural defects. To differentiate these two states, sperm viability assays are used. Traditional viability assays expose sperm to dyes that may penetrate dead sperm but are excluded from live sperm with intact cell membranes. Eosin Y and trypan blue stains are commonly employed. Dead sperm are stained, and live sperm remain unstained. This assay is indicated in samples with absent motility or very low (>10%) motility.

The hypo-osmotic sperm-swelling test is based on the principle that live sperm with intact membranes will be able to maintain an osmotic gradient. When placed in a hypo-osmotic solution, water will flow into viable cells, causing the membrane to bulge, which is particularly noticeable in the sperm tails. Nonviable sperm will not maintain an osmotic gradient and therefore will not swell (Jeyendran et al, 1984). The results of this assay generally correlate well with the results of standard sperm viability staining (Avery et al, 1990; Jeyendran et al, 1992).

There have been conflicting reports on the use of this assay in predicting IVF rates as well as the correlation of this assay with other tests of fertility (Chan et al, 1990). As a result, this test has not gained widespread use as a diagnostic test of male infertility.

More recently, the hypo-osmotic sperm swelling test has been useful in identifying and selecting viable sperm to be injected during IVF with ICSI when the sperm are nonmotile. In these instances, sperm that demonstrate swelling under hypo-osmotic conditions are viable and may be used for ICSI (Casper et al, 1996; Barros et al, 1997; Liu et al, 1997).

Reactive Oxygen Species Testing

Under aerobic conditions, human cells produce various reactive oxygen species (ROS) such as superoxide radicals (O2- ), hydrogen peroxide (H2O2), and the hydroxyl radical (OH- ). Although these metabolites may play a normal physiologic role in processes such as sperm hyperactivation and capacitation (de Lamirande and Gagnon, 1993), they may also be toxic to sperm. These ROS induce peroxidative damage to cell lipid membranes. Detrimental effects on sperm metabolism, morphology, motility, and fertilizing capacity have been demonstrated (Alvarez and Storey, 1982; Aitken and Clarkson, 1987; Aitken et al, 1989; Rao et al, 1989). Both spermatozoa and white blood cells in semen produce ROS, although white blood cells produce far more than do sperm. A greater proportion of infertile men demonstrate elevated levels of seminal ROS as compared with populations of fertile men (Iwasaki and Gagnon, 1992; Mazzilli et al, 1994). Although there is clear evidence that ROS are detrimental to sperm function, the indications for ROS testing remain unclear. Some believe that abnormal ROS levels are the cause for some forms of unexplained infertility, but it may often be a result of infertility (Conte et al, 1999). Currently, ROS testing has not yet become routinely available.

Genetic Testing

Genetic causes for male infertility include karyotypic abnormalities (structural or numerical chromosomal abnormalities), Y chromosome microdeletions, and autosomal gene mutations. Chromosomal abnormalities may involve large or small amounts of chromatin. The karyotype analysis detects numerical and structural chromosome abnormalities involving large amounts of DNA. Very small deletions will not be identified by this technique but require microdeletion analysis. Approximately 6% of infertile men are found to have chromosome abnormalities detected by karyotype analysis. The prevalence of abnormalities increases as the sperm count decreases. The highest prevalence is found in azoospermic patients, with 10% to 15% of patients demonstrating abnormalities of the karyotype (Hendry et al, 1975; Chandley, 1979; Retief et al, 1984; Bourrouillou et al, 1985, 1992). The prevalence falls to 4% to 5% in oligospermic patients and 1% in normospermic patients (Matsuda et al, 1992). This is significantly higher than the approximately 0.4% prevalence of chromosome abnormalities in newborns. Sex chromosomal abnormalities predominate in azoospermic men, and autosomal abnormalities predominate in oligospermic men.

Y chromosome microdeletions of sections of the long arm of the Y chromosome have been identified in approximately 13% of azoospermic men and 3% to 7% of oligospermic men (Reijo et al, 1995; Nakahori et al, 1996; Seifer et al, 1999). Approximately 7% of unselected infertile men have been found to have Y chromosome microdeletions (Girardi et al, 1997; Pryor et al, 1997; Kleiman et al, 1999). Y chromosome microdeletion analysis is not universally available nor is the technique standardized at the present time. Currently, genetic causes of male infertility are not curable; however, many of these patients are candidates for IVF combined with ICSI. Because of this, patients who are considered candidates for the ARTs and with severe oligospermia (>5 million sperm/ml) or nonobstructive azoospermia should be offered genetic screening with both karyotype and Y chromosome microdeletion analysis.

Testicular Biopsy

Diagnostictesticular biopsy is performed only on azoospermic patients. Most clinicians perform bilateral testicular biopsy, but, in patients with discrepant testicular volume, some physicians perform a biopsy on the larger testis only. The purpose of a diagnostic testicular biopsy is to differentiate between obstructive and nonobstructive azoospermia. Patients with clinical findings that are pathognomonic for either obstruction, such as CBAVD, or testicular failure do not require a testicular biopsy to establish the cause of azoospermia. Clinical findings pathognomonic for testicular failure include bilateral small testes and a markedly elevated serum FSH. Thus, a diagnostic testicular biopsy is needed only in those patients in whom ductal obstruction is suspected based on the presence of a relatively normal serum FSH and testicular volume (Jarow et al, 1989).

The other role of testicular biopsy is in the management of patients with nonobstructive azoospermia who are being considered candidates for sperm retrieval and IVF. In this setting, a testicular biopsy may be performed either to obtain prognostic information or to harvest sperm for cryopreservation. Many patients with nonobstructive azoospermia have limited numbers of spermatozoa that may be harvested from the testis and used in vitro to fertilize eggs (Devroey et al, 1995). Therefore, one might consider the possibility of cryopreserving sperm for later use in IVF in patients undergoing a diagnostic testicular biopsy. Testicular biopsy is not indicated in patients with oligospermia, because the results will not alter therapy. A biopsy is rarely performed to rule out partial ductal obstruction in patients with severe oligospermia, normal-sized testes, and normal FSH values. Partial ductal obstruction is suggested in these cases if the biopsy specimen demonstrates normal spermatogenesis. The details of the technique of testicular biopsy are discussed at length in Chapter 44, Surgical Management of Male Infertility and Other Scrotal Disorders.

The interpretation of testicular biopsies is subjective and suffers from a lack of uniformity of the systems of classification. Objective methods to quantify spermatogenesis are reproducible but rarely add to the clinical management and are thus used primarily in research studies (Johnsen, 1970; Silber and Rodriguez-Rigau, 1981). The most commonly employed classification patterns are based on the appearance of spermatogenesis, ranging from normal to Sertoli cell only with maturation arrest and hypospermatogenesis in between. The examination should evaluate the size and number of seminiferous tubules, the thickness of the seminiferous tubule basement membrane, the relative number and types of germ cells within the seminiferous tubules, the degree of fibrosis in the interstitium, and the presence and condition of Leydig cells. Very commonly, more than one pattern is identified in a single biopsy specimen. This has contributed to some of the inconsistencies in classification systems. The following classification scheme is commonly used and clinically practical.

Normal Testes

The bulk of the volume of the normal testis is made up of seminiferous tubules that are separated by a thin layer of loose interstitium containing Leydig cells, blood vessels, lymphatics, and connective tissue ( Fig. 43–9). Leydig cells are acidophilic, are round to polygonal cells found in groups, and may contain crystalloids of Reinke. Sertoli cells and spermatogonia line the basement membrane of the seminiferous tubule. The steps of spermatogenesis include mitotic division of the stem cells (spermatogonia), meiotic division of the germ cells (primary and secondary spermatocytes), and spermiogenesis or the development of a spermatozoon (spermatids). Germ cells in all steps of spermatogenesis should be seen within the seminiferous tubules. However, not all tubules contain all stages of spermatogenesis. Unlike most other mammalian testes that exhibit the stages of spermatogenesis in a wave along the tubule, the human has a patchwork pattern. Normal testicular biopsy specimens are found in azoospermic patients with ductal obstruction. However, before there is distal obstruction, overcrowding of the tubule lumens and disorganization are common (Levin, 1979). In addition, the seminiferous tubules become dilated and their walls thickened with long-term obstruction (Jarow et al, 1985).

Hypospermatogenesis

A reduction in the number of all germinal elements within the seminiferous tubule is present in cases of hypospermatogenesis. Thus, histologic examination reveals thinner layers of germ cells within the seminiferous tubules. The organization of the germinal epithelium may be disrupted, and immature germ cells may be found in the lumen in some instances. The interstitium and Leydig cells are normal. Patients with hypospermatogenesis often have oligospermia but, in severe cases, may be azoospermic. A certain level of sperm production must be reached before sperm are seen in the ejaculate.

Maturation Arrest

Histologic examination of these testes reveals spermatogenesis proceeding normally through a specific stage at which point no further maturation of germ cells is identified. The arrest may occur at the primary spermatocyte, secondary spermatocyte, or spermatid stage. In a given patient, the block is at a consistent stage. Cases of late maturation arrest are often difficult to differentiate from normal spermatogenesis without the use of a testicular touch preparation (Kim et al, 1996). Mature spermatozoa are present on a testicular touch preparation in patients with normal spermatogenesis and absent in cases of complete late maturation arrest. Patients with complete maturation arrest at any stage exhibit azoospermia, whereas patients with partial maturation arrest have varying degrees of oligospermia. It is common to see a mixture of maturation arrest and hypospermatogenesis in the same testis.

Germinal Aplasia

Germinal aplasia is also known as Sertoli cell–only syndrome. Testicular histology reveals seminiferous tubules containing Sertoli cells with a complete absence of all germ cells. The diameter of the seminiferous tubules is reduced, and the interstitium is usually minimally altered. Patients with Sertoli cell–only syndrome have small to normal-sized testes associated with normal or elevated levels of FSH (Turek et al, 1995). There is no effective treatment for this condition. However, many patients with a Sertoli cell–only pattern on a diagnostic biopsy have low levels of spermatogenesis in other areas of the testis. This condition should also be differentiated from end-stage testes, which is a sclerotic testis with some tubules containing only Sertoli cells.

End-Stage Testes

Tubular and peritubular sclerosis is characteristic of end-stage testes. Germ cells are absent from the sclerotic seminiferous tubules. Sertoli cells may or may not be present. Leydig cells may be absent or decreased in number within the sclerotic interstitium. Clinically, these testes are bilaterally atrophic and firm. A gradual decrease in spermatogenic activity leads to a reduction and disappearance of all germ and Sertoli cells in the testes of patients with Klinefelter’s syndrome. Tubule sclerosis and hyalinization usually result. Clumping of Leydig cells may be apparent.

Other Patterns

In cases of hypogonadotropic hypogonadism, seminiferous tubules remain very small, demonstrating an absence of germ cells and Leydig cells. This is the pattern of a 7-month-old fetus. In cases of isolated LH deficiency (fertile eunuchs), normal spermatogenesis or hypospermatogenesis may be present. However, Leydig cell numbers may be reduced. Testicular atrophy, manifested as normal-sized seminiferous tubules with a depletion of germ cells, is found in hypophysectomized adult males who were sexually mature at the time of gonadotropin depletion. Leydig cells may be depleted or absent in these specimens.

The testicular biopsy is rarely pathognomonic of a single etiology. The most common abnormalities in infertile men are hypospermatogenesis and maturation arrest. In addition, several patterns may be present in an individual biopsy specimen. Thus, in most cases, a testicular biopsy does not result in the identification of a specific etiologic factor of a patient’s infertility.

Percutaneous fine-needle aspiration cytology has been used to determine the presence of spermatogenesis (Cohen et al, 1984). This technique may be performed in the office without anesthesia but requires a highly skilled cytologist. The interpretation is similar to that of a testicular touch preparation. Flow cytometry may be combined with fine-needle aspiration and the ploidy patterns correlated with the state of spermatogenesis (Chan et al, 1984; Kaufman and Nagler, 1987). Although published clinical studies have documented the efficacy of these techniques, the requirement for specialized skills or equipment has limited their popularity.