Collection and Timing

Physicians should provide patients with standard guidelines for the collection of semen because suboptimal sperm collection remains a frequent cause of error in semen analysis. There should be 2 to 7 days of sexual abstinence before collection. Two separate samples at least 7 days apart should be analyzed (Rowe, 2000; Jeyendran, 2003). The duration of abstinence should be constant, if possible, because each additional day can add as much as 25% in sperm concentration (Carlsen et al, 2004). Lubricants should be avoided because they may interfere with motility results. Coitus interruptus should be discouraged because it often leads to inaccurate results (i.e., the first part of the ejaculate, which contains most of the sperm, may be lost).

Masturbation in a clinical setting is the recommended procedure. Collection is done in a private room in the same facility where the semen will be analyzed. The glans and the penis should be cleaned with a wet paper towel (soap should be avoided). Lubricant use is discouraged but, if necessary, should not be applied to the glans. A clean, sterile container should be used for specimen collection. The container should be provided by the laboratory to avoid contamination or spermicidal effects. The main advantages of this collection method are its simplicity, noninvasiveness, and inexpensiveness (Jeyendran, 2003).

Some men may not be able to achieve adequate erection and ejaculation. Assistance can be provided to them by oral medications such as phosphodiesterase type 5 inhibitors given 30 to 60 minutes before collection. Cavernosal and subcutaneous injections of prostaglandins are less popular but remain possible options for patients who have erectile dysfunction. Seminal pouches that do not contain any spermicides allow the patient to engage in sexual activity if he is incapable of or uncomfortable producing specimens by masturbation. Vacuum erection devices can also be used to obtain erection by creating a vacuum around the penis, generating a pressure differential that fills the corpora with blood. Vibratory stimulation may be used for patients who have suffered spinal cord injury, if the spinal cord lesion is T8 and above (Brown et al, 2006). Rectal probe electro-stimulation induces ejaculation by stimulation of the efferent fibers of the hypogastric plexus. Precautions for autonomic dysreflexia should be taken while doing these procedures because some patients with high spinal cord lesions (T6 and above) can have life-threatening hypertension (Jeyendran, 2003).

In order to allow liquefaction and mixing, semen is placed in a 37° C gently shaking incubator for 30 minutes. The semen sample should be examined within 1 hour of production and receipt in the laboratory. Some of the semen parameters can be affected by a delay in assessment. Motility decreases significantly after 2 hours and progressively diminishes afterwards as free radical activity increases.

The semen analysis characteristics can be classified into two groups: macroscopic and microscopic.

Macroscopic Assessment

The five macroscopic measurements in a standard sperm analysis have remained fairly constant, with the normal values remaining relatively unchanged since the inception of the semen analysis in the 1950s (Table 21–2). Normal human semen is an off-white to grayish-yellow opalescent fluid. In event of urine contamination, the semen sample has a yellow discoloration. The semen may appear pink in patients with urethral bleeding and yellowish in jaundice patients. During the time of ejaculation, the spermatozoa are suspended in the secretions of prostate, seminal vesicles, bulbo-urethral glands, and other accessory glands that form a coagulum. The specimen usually liquefies within 30 minutes. However, semen obtained from patients with congenital bilateral absence of the vas usually does not form a coagulum and is acidic. Liquefaction is aided by the proteolytic enzyme fibrinolysin, secreted by the prostate. Improper or prolonged liquefaction indicates an ejaculatory duct obstruction or poor prostatic secretion. Viscosity and nonliquefaction are two different phenomena often confused. Viscosity relates to the fluid nature of the sample. It is measured by dropping the semen sample into a container using a pipette and observing the length of the thread formed. Increased viscosity is often associated with infertility because it is known to impair sperm movement. Semen samples that are highly viscous can be treated with enzymes such as trypsin before they are processed for therapeutic purposes. Measurement of pH is a standard component of semen analysis and is largely determined by the secretions from the seminal vesicles and the prostate. The normal range of pH has been defined as 7.2 to 8.0. Because the secretions of seminal vesicles are alkaline, acidic pH indicates congenital absence of the vas with the associated seminal vesicle hypoplasia seen in azoospermic patients (WHO, 1999).

Table 21–2 Macroscopic Features of Semen Analysis

Microscopic Assessment

Morphology

Sperm morphology is the most subjective and most difficult-to-standardize semen parameter. Accurate assessment of morphology is critical in the evaluation of an infertile male because it can be a significant predictor of pregnancy. Normal sperm possess an oval head with a well-defined acrosomal region composing 40% to 70% of the head area. The dimensions of the head are 4 to 5.5 µm in length and 2.5 to 3.5 µm in width. The normal sperm are free from head, midpiece, or tail defects. Head defects include microcephalic head (approximately half the size of a normal sperm head), megalocephalic head (one-and-a-half times the size of a normal sperm head), tapered head, round sperm (missing acrosome), and bicephalic or multicephalic head. Neck defects include no tail or improper tail insertions. Midpiece defects comprise elongated, distended, thin, or bent midpieces. Some of the tail defects commonly noted are short, multiple, bent, or broken tails. One common defect includes coiled tail, which is indicative of osmotic stress (McLachlan, 2003).

Sperm morphology is expressed as percentage of abnormal forms present in the semen. The two most common classifications used for the assessment of sperm morphology are the WHO criteria and Kruger’s strict criteria (Table 21–3). When correctable causes of male infertility are not identified, couples with teratozoospermia (<15% normal morphology by WHO method) may be directed to proceed with IVF and ICSI as compared with intrauterine insemination (IUI). Teratozoospermia may occur due to several factors such as fever, varicocele, and stress. Some drugs that affect spermatogenesis are also known to cause morphologic abnormalities. With the advent of ICSI, which requires only one morphologically and functionally normal spermatozoa to fertilize an oocyte, morphologic assessment is losing its significance (Zinaman, 2000).

Table 21–3 Sperm Morphology Classification

	WORLD HEALTH ORGANIZATION 3RD	STRICT WORLD HEALTH ORGANIZATION 4TH (KRUGER)
Normal reference range	>30%	>14%
Head
Shape	Oval	Oval, smooth borders
Acrosome	40%-70% of head surface	40%-70% head surface
Size	4-5.5 mm length 2.5-3.5 mm width Length/width 1.5-1.75	3-5 mm length 2-3 mm width
Vacuoles	<20% head area	Up to 4
Midpiece
Shape	Straight regular outlined Axially arched	Slender, straight, regular outline Axially arched
Size	< of head area	<1 mm wide Length 1.5 × head
Cytoplasmic droplet	< of head area	< of head area
Tail
Appearance	Slender, uncoiled	Uniform, uncoiled
Width		Thinner than midpiece
Length	>45 mm	10 × head

Computer-Assisted Sperm Analysis

Computer-assisted sperm analysis (CASA) is a semiautomated technique that provides data on sperm density, motility, straight-line and curvilinear velocity, linearity, average path velocity, amplitude of lateral head displacement, flagellar beat frequency, and hyperactivation. It has two distinct advantages over traditional manual analyses: high precision and quantitative assessment of sperm kinematics. Sperm concentration, samplepreparation, and frame rate can, however, affect accuracy of the CASA (Mortimer, 1994). The use of some stains has also affected the accuracy of determining the sperm morphology. Although this technology has theoretic advantages, it has not translated into benefits in clinical practice. This test requires expensive equipment and still requires the active participation of a technician. Therefore at present, these machines are found commonly in andrology laboratories, not in general pathology laboratories, where most of the initial semen analyses are analyzed (Amann and Katz, 2004). Presently, the most important role of CASA is to provide standardized aids in quality control and quality assurance in andrology laboratories, as the emerging use of ICSI has diminished the role of motility assessment in sperm selection (Amann and Katz, 2004).

Limitations of Semen Analysis

The true litmus test for male fertility remains the ability to cause pregnancy in vivo. Although the semen analysis is used as a surrogate measure of a man’s fertility potential, it is not a direct measure by any means. Clinical research has shown that normal semen analysis may not reflect defects in sperm function (idiopathic infertility), and men with poor sperm parameters still may cause spontaneous pregnancies. Only 50% of infertile men have recognizable causes detectable by the basic semen analysis (MacLachlan, 2003). The presence of several criteria further reinforces the emerging opinion that the current standards do not reflect the true fertility potential of subjects. The current normal values fail to satisfy clinical and statistical standards (McLachlan, 2003; Nallella et al, 2006) and pose the risk of misclassifying a subject’s true fertility status. In fact, 20% of 18-year-olds would be classified as subfertile using the WHO cutoff of 20 × 10⁶ sperm/mL (Andersen et al, 2000). Studies on semen donors with known fertility status have revealed a significant overlap in the sperm characteristics between fertile and subfertile men (Li et al, 2006; Nallella et al, 2006). Guzick and colleagues (2001) in a study of 1461 men found different cutoff levels in sperm concentration (<13.5 × 10⁶ in subfertile and 48 × 10⁶ in fertile men), percent motility (<32% in subfertile and >63% in fertile men), and normal morphology (<9% in subfertile and >12% in fertile men). Nallella and colleagues in 2006 did a similar study (n = 572) and used the WHO and Tygerberg criteria on subjects with known fertility. They noted that there is low sensitivity (0.48) in detecting subfertile subjects using WHO reference values for sperm concentration and low sensitivity (0.83) using Tygerberg criteria for percentage of normal morphology. Among the variables, motility had the least overlap range and gave the best prediction of the subject’s fertility potential. This is in contrast with the earlier study by Guzick and colleagues, where morphology was reported to provide the highest discriminating power in detecting subfertility among all the semen variables. Clearly, each variable alone is neither a powerful sole discriminator nor a predictor of fertility status, and they must be considered in the context of other parameters and the clinical setting. There remains a need for further studies in larger populations and different demographics before a consensus can be reached on the necessity of resetting current values to increase the predictiveness and utility of the semen analysis (Table 21–4).

Table 21–4 Characteristics of Normal Semen (World Health Organization, 1999)

Sperm-Mucus Interaction/Postcoital Test

Cervical mucus is a heterogenous fluid that is composed of 90% water. In order to reach the site of fertilization, the spermatozoon must be able to successfully traverse the cervix and the cervical mucus. In-vitro penetration of spermatozoa through cervical mucus is comparable to in-vivo conditions. The cervical mucus is shown to demonstrate cyclical changes in consistency and to be highly receptive around the time of ovulation. Increase in penetrability is often observed one day before the LH surge. Cervical mucus has been shown to protect the spermatozoa from the hostile environment of the vagina. The penetrability of the spermatozoa through the cervical mucus can be detected by the cervical mucus migration assay. Some methods by which migration can be detected include the postcoital test (PCT). This test can assess cervical environment as a cause of infertility. Accurate timing is crucial because it must be conducted when the cervical mucus is thin and clear just before ovulation. In this test, cervical mucus is examined 2 to 8 hours after normal intercourse. Progressively motile sperm greater than 10 to 20 per HPF is designated as normal. Practical guidelines of the American Society of Reproductive Medicine recommend PCT in the setting of hyperviscous semen, unexplained infertility, or low-volume semen with normal sperm count (Van der Steeg et al, 2004). Medical history and semen analysis can predict PCT results in half of the infertile couples. Poor-quality semen most likely will have poor PCT. Therefore it is not recommended routinely for men who have abnormal semen analyses. Couples who show defective sperm mucus interaction may be advised to proceed with IUI because additional tests are unlikely to affect the management (Guzick et al, 2001). However, abnormal PCT may result from inappropriate timing of the test. Other causes of abnormal PCT include anatomic abnormalities, semen or cervical mucus antisperm antibodies, inappropriately performed intercourse, and abnormal semen. Persistently abnormal PCT in the presence of reasonably good semen parameters should indicate poor cervical mucus quality. The finding of good-quality mucus with nonmotile spermatozoa or immobilized sperm demonstrating shaking motion should lead to the evaluation of both partners for the presence of antisperm antibodies. Although it has fallen out of favor, this test may be useful in patients who are unable or unwilling to produce an ejaculate.

Acrosome Reaction

The Acrosome is a membrane-bound organelle that covers the anterior two thirds of the sperm head. Acrosome reaction is an important prerequisite for successful fertilization. It is an exocytotic event that involves fusion of outer acrosomal membrane and sperm plasma membrane, which enables the exposure of acrosomal contents through the formation of vesicles. The two important acrosomal enzymes that are required to digest the oocyte cumulus cells and zona pellucida include acrosin and hyaluronidase. Acrosome reaction testing is not widely practiced in laboratories and only remains a research interest. However, this test may be recommended in cases of profound abnormalities of head morphology or in the setting of unexplained fertility in patients with poor IVF pregnancy rates. Normal semen samples demonstrate spontaneous acrosome reaction rates of less than 5% and induced acrosome reaction rates of 15% to 40%. Infertile populations have shown high spontaneous rates of acrosome-reacted sperm and low rates of induced acrosome reactions. Although not widely practiced due to its cost and labor, the structure of the acrosome can be studied under transmission electron microscopy. Other techniques such as fluorescence microscopy and beads coated with antiacrosomal antibodies have been developed, but these tests are also not readily available in standard laboratories.

Sperm Penetration Assays/Sperm Zona Binding Tests

The sperm penetration assay (SPA) or the hamster egg penetration assay (HEPT) determines the functional capacity of the spermatozoa necessary to fertilize an oocyte. It is based on the principle that normal spermatozoa can bind and penetrate the oocyte membrane, which is a prerequisite for the fusion of sperm and the oocyte. Zona pellucida is the outermost layer protecting the cytoplasm of the oocyte. It plays an important role in the fertilization process and is shown to be the only physiologic inducer of acrosome reaction. Sperm binds to the species-specific receptor, ZP3, which is found on the zona pellucida of the oocyte. The zona-free hamster oocytes, in which the zona pellucida is stripped, are used to allow cross-species fertilization. Human sperm penetration assay with zona-free hamster eggs determines the ability of sperm to successfully undergo capacitation, acrosome reaction, membrane fusion with oocytes, and chromatin decondensation. The assay is performed by incubating zona-free hamster oocytes in sperm droplets for 1 to 2 hours. The oocytes are examined microscopically for sperm penetration. Penetrations are indicated by swollen sperm heads within the oocyte cytoplasm. Normally, 10% to 30% of ova are penetrated (WHO, 1999). Oligozoospermic and severely teratospermic men have a higher number of defective sperm-zona pellucida interactions, which may account for their low fertility potential in both spontaneous and IVF pregnancies (Liu and Baker, 2004). Despite its low predictive power, SPA is correlated positively with spontaneous pregnancy outcomes (Corson et al, 1988). Sperm capacitation index (SCI) is a variant of the SPA test, assessing the mean number of penetrations per ovum. ICSI has been recommended for couples with an SCI less than 5 instead of standard IVF procedures (Ombelet et al, 1997). Compared with SPA, the zona binding test uses oocytes that failed to fertilize in IVF clinics. The need for human oocyte supply, however, remains a limitation to the use of this test.

Antisperm Antibody Testing

The tight Sertoli-cell junctions provide the testis with a barrier that prevents the immune system from coming in contact with the post-meiotic germ cells. However, in certain conditions such as testicular torsion, vasectomy, and testicular trauma, this unique barrier can be violated, resulting in an immune response to sperm, displayed as antisperm antibodies (ASABs). These antisperm antibodies can be several types—sperm agglutinating, sperm immobilizing, or spermotoxic. The sperm agglutinating type causes agglutination of spermatozoa, which reduces the availability of motile spermatozoa penetrating the cervical mucus. Sperm-immobilizing antibodies induce loss in motility of the sperm, which can be identified by the characteristic “shaking” pattern in motility on postcoital test. The spermotoxic type of ASAB causes a complement-dependent loss in viability of spermatozoa.

Approximately 10% of infertile men will present with ASA as compared with 2% of fertile men (Guzick et al, 2001). Sperm parameters are often normal in men with ASA (Munuce et al, 2000). Hence it has been suggested to be tested routinely in all men undergoing infertility work-ups (McLachlan, 2003). Excessive sperm agglutination or an abnormal PCT can suggest the presence of ASA.

The direct ASA test detects sperm-bound immunoglobulins. Indirect testing detects the biologic activity of circulating ASA. False positives can result from nonimmunologic factors (Francavilla et al, 2007). Because only antibodies present on the sperm surface are clinically significant, most investigators prefer direct assays that determine sperm-bound antibodies instead of indirect detection of serum antisperm antibodies. IgG-MAR (mixed antiglobulin reaction) and Sperm MAR are recommended screening tests that are economical and readily available. Immunobead Test (IBT), which measures IgG, IgA, and IgM, may be additionally recommended when either of the previous tests gives a positive result in order to determine if IgA are bound to sperm surface. Acceptable normal values by WHO (1992) standards include less than 10% (IgG MAR) or 20% (IBT) of spermatozoa with adherent particles.

Clinical implications of ASA on male infertility are varied. A weakly positive IgG MAR/IBT in men who have low motile sperm rules out immunologic factors, and no further testing is necessary (Francavilla et al, 2007). ASA are present in 34% to 74% of vasectomized men and persist in 38% to 60% after vasectomy reversal (Broderick et al, 1989; Francavilla et al, 2007). Routine ASA testing is not recommended in this setting because it is of uncertain significance and usually does not affect the decision to do a vasectomy reversal. There are conflicting reports regarding ASA levels after orchidopexy for cryptorchidism (Mirilas et al, 2003). In genitourinary infections, ASA is thought to be a consequence of the inflammatory process rather than cross reactivity to the microorganism (Francavilla et al, 2007).

The decision to proceed with IUI versus ICSI in immunologic infertility can be aided by a zona pellucida (ZP) test. If the sperm exhibit inability for ZP binding, ICSI is the procedure of choice. Presently, flow cytometry techniques are being developed to quantify ASA in individual spermatozoa (Shai et al, 2005). These techniques are also being explored to identify sperm surface antigens for possible immunocontraceptive development.

Electron Microscopy

Spermatozoa may test positive for viability even in the presence of ultrastructural defects. Ultrastructural details of the sperm can only be seen under the electron microscope (EM). Patients who have low sperm motility (<5% to 10%) with high viability (as determined by HOST or Eosin-Nigrosin staining) and density may be appropriate candidates for EM assessment. Subfertile men may demonstrate more serrated and blurred circular sulcus, less intact acrosome membrane, a bigger proportion of the spermatic head, and more droplets attached to the acrosome membrane. Mitochondrial and microtubular defects that are not visible under the usual Papanicolaou smear can be detected.

Biochemical Tests

Acrosin is a serine protease-like enzyme that exhibits a lectin-like carbohydrate binding activity to the zona pellucida glycoproteins. Low acrosin activity has been associated with low sperm density, motility, and poor normal morphology (Xu and Zhan, 2006).

Zinc is necessary for chromatin stability and decondensation, as well as for head–tail detachment during fertilization. It is measured by colorimetric methods with a reference value of 13 mmoL per ejaculate (WHO, 1999). Reports on the effects of zinc in sperm function and semen parameters are quite conflicting. Mankad and colleagues (2006) reported positive correlations between seminal zinc levels, alpha glucosidase, and sperm count; however, there are other reports that showed no significant changes in sperm count and motility with variations in zinc concentration (Abou-Shakra et al, 1989; Lewis-Jones et al, 1996; Sorensen et al, 1999). Zinc levels in seminal plasma are decreased, but spermatozoal zinc levels are increased in asthenozoospermic and oligoasthenozoospermic men (Zhao and Xiong, 2005). A low zinc-to-calcium ratio has been shown to be associated with better motility than high ratio (Sorensen et al, 1999). However, dietary supplementation of zinc did not improve semen variables (Agarwal and Said, 2004).

The seminal vesicles contribute to the bulk of seminal fluid that serves as the transport medium for sperm and contribute to the nutrition in the form of fructose. There is a positive correlation between sperm motility and seminal fructose levels (Lewis-Jones, 1996). Low or absent fructose is seen in ductal obstruction and congenital conditions like CBAVD. Semen fructose testing may be requested when hypo-functioning seminal vesicles are suspected, although morphometric analysis of seminal vesicles using transrectal ultrasound (TRUS) is the recommended test nowadays.

L-carnitine is secreted by the epididymis and is concentrated in the seminal plasma at up to 10 times the serum levels. It has a role in sperm maturation. Low L-carnitine levels are found in oligoasthenozoospermic men (Agarwal and Said, 2004; Sigman et al, 2006). The levels of carnitine can possibly serve as indicators of the level of obstruction in the ductal system. Extremely low concentrations of L-carnitine are found in azoospermic men who have postepididymal obstructions, whereas normal levels are found in azoospermic men who have intratesticular obstructions (Agarwal and Said, 2004). Administration of L-carnitine supplements did not improve sperm density, but contrasting results have been reported for sperm motility changes (Sigman et al, 2006). L-carnitine determinations remain far from becoming mainstream tests in male infertility until significant well-designed studies are conducted.

Alpha glucosidase, tested by fluorimetric methods, has been used to distinguish nonobstructive from obstructive azoospermia. It is used as a specific marker for epididymal function and is believed to play a role in sperm maturation in the epididymis. A cutoff value of 12 mIU/mL distinguishes ductal obstruction from primary testicular failure (Comhaire et al, 2002). The usefulness of this test was questioned by Krause and Bohring (1999), but Comhaire and colleagues (2002), in their review, showed a strong association between α-glucosidase and semen parameters. The cutoff level had 95% specificity in identifying obstructive azoospermia. This suggests that the test can predict IUI response (higher pregnancy rate >78 U per ejaculate) because high levels indicate better zona-binding capacity (Comhaire et al, 2002). The presence of commercial test kits using colorimetric methods promises to make testing accessible and affordable.

Reactive Oxygen Species

Research conducted during the past decade has provided growing support for the concept that excessive production of reactive oxygen species (ROS) is related to abnormal semen parameters and sperm damage. Routine semen analysis remains the backbone of clinical evaluation in male infertility, and determining the levels and sources of excessive ROS generation in semen is currently not included in the routine evaluation of subfertile men. However, the diagnostic and prognostic capabilities of seminal oxidative stress measurement exceed the capabilities of conventional sperm quality tests. An oxidative stress test may accurately discriminate between fertile and infertile men and identify those with a clinical diagnosis of male factor infertility who are likely to initiate a pregnancy if they are followed over a period of time. In addition, such a test can help select subgroups of patients with infertility in which oxidative stress is an important factor and who may benefit from antioxidant supplementation. Although consensus is still required about the type and dosage of antioxidants to be used, rationale and evidence exist supporting their use in infertile men with elevated oxidative stress (Deepinder et al, 2008).

Currently, clinical practice as to the inclusion of ROS measurement is variable, primarily because of the lack of standardization of ROS analytic methods, equipment, and range of normal levels of ROS in semen. The evidence defining high ROS levels as a cause or an effect of abnormal semen parameters and sperm damage is still insufficient on both sides of the question. However, it has been reported that a high level of ROS is an independent marker of male factor infertility in leukocytospermic samples after adjustment for semen characteristics. This finding suggests that ROS may play an important role in the etiology of male factor infertility and encourages the use of ROS measurement as a diagnostic tool in clinical practice, particularly in cases of idiopathic infertility. Although numerous assays for ROS measurement have been introduced, the chemiluminescence assay, determining ROS levels in neat semen, has proven to be an accurate and reliable test for evaluating oxidative stress status. This technique accurately represents an individual’s true in vivo oxidative stress status and overcomes the drawbacks of earlier methods that involve processing semen, a step that may generate ROS by itself. The ROS level for healthy donors with normal standard semen parameters is 1.5 × 10⁴ cpm/20 million sperm/mL. Using this value as a cutoff, infertile men can be classified as either oxidative stress positive (>1.5 × 10⁴ cpm/20 million sperm/mL) or oxidative stress negative (≤1.5 × 10⁴ cpm/20 million sperm/mL), regardless of their clinical diagnosis or standard semen analysis results (Deepinder, et al, 2008).

Sperm DNA Damage

DNA fragmentation was initially described in 1993 and has since been researched as a test to aid fertility prediction in subfertile males. The spermatozoal chromatin is a tightly packed structure because of the disulfide cross linkages between protamines that allow compaction of the nuclear head and protect the DNA fragments from stress and breakage. DNA damage is multifactorial and theories on its etiology include protamine deficiency and mutations that may affect DNA packaging or compaction during spermiogenesis (Agarwal and Said, 2003). Various factors found to be associated with increased sperm DNA damage include tobacco use, chemotherapy, testicular carcinoma, and other systemic cancers (Agarwal and Said, 2003). DNA damage is correlated positively with poor semen parameters, especially low sperm concentration and low sperm motility, leukocytospermia, and oxidative stress (Erenpreiss et al, 2002; Agarwal and Said, 2003; Zini and Libman, 2006). Approximately 8% of subfertile men who have normal semen parameters will have high abnormal DNA (Aitken et al, 1991).

Many tests of sperm DNA damage are now available (Table 21–5). The use of these tests has been driven largely by the growing use of assistive reproductive technologies and awareness that the integrity of the male genome plays an important role in IVF. Sperm DNA damage can be measured directly (fragmentation, oxidation) or indirectly (sperm chromatin compaction). Direct assessment of DNA damage can be obtained by means of single-cell gel electrophoresis assay or “comet” assay (electrophoresis causes DNA fragments to migrate away from the central DNA core, revealing a “comet”), terminal deoxynucleotidyl transferase-mediated dUTP-nick end-labeling or “TUNEL” assay (the ends of fragmented DNA are tagged), and liquid chromatography to measure DNA oxidation levels. DNA damage can also be assessed indirectly by means of sperm chromatin integrity assays and by evaluation of nuclear protein levels. Sperm chromatin integrity assays include slide-based sperm nuclear protein stains (e.g., aniline or toludine blue [detects histones], CMA3 [detects underprotamination]) and DNA stains (e.g., acridine orange [detects denatured or single-stranded DNA]). The sperm chromatin structure assay (SCSA) uses flow cytometry to estimate the percentage of spermatozoa with DNA denaturation (spermatozoa are stained with acridine orange). A cutoff rate of greater than 30% has been shown to be associated with a significant decrease in in-vivo fertilization rates (Evenson and Wixon, 2002). A DNA fragmentation index (DFI) of greater than 30% has a sensitivity of 15% and a specificity of 96%. Meta-analyses by Evenson and Wixon (2002) and Li and colleagues (2006) showed that couples are twice as likely to become pregnant with regular IVF methods if the DFI is less than 30%. Contrasting reports, however, have failed to show significant correlation between DNA damage and idiopathic infertility (Verit et al, 2006). In addition, significant intraindividual variation exists making conclusions using SCSA problematic (Erenpreiss et al, 2006). There is a higher rate of DNA damage in ejaculated or epididymal sperm than in intratesticular spermatozoa. Hence the use of intratesticular spermatozoa from high DFI men is recommended for ICSI (Steele et al, 1999; Greco et al, 2005). ICSI is advised when DFI is above cutoff levels. DNA fragmentation testing can help couples decide on what fertility modality and possible lifestyle modifications they can employ to increase their chances of conception.

Table 21–5 Commonly Used Tests of Sperm DNA Damage

Transrectal Ultrasonography

TRUS provides excellent definition of the prostate, seminal vesicles, ampulla of the vas deferens, and the ejaculatory ducts. TRUS is primarily employed to examine patients suspected to have ejaculatory duct obstruction (EDO). These patients usually have low-volume azoospermia (volume <1 mL) with acidic pH and negative semen fructose. TRUS typically employs the 5- to 7-MHz endocavitary probe with scanning in both the longitudinal and transverse planes. Careful examination of verumontanum may identify midline prostatic cysts such as müllerian or wolffian duct cysts or stones obstructing the ejaculatory duct (Fig. 21–2). Often the ejaculatory duct may not be well visualized, but dilation of the seminal vesicles serves as a de facto sign of ejaculatory duct obstruction. Although not always present with ejaculatory duct obstruction, seminal vesicle width in excess of at least 12 to 15 mm or ejaculatory duct diameter greater than 2.3 mm is considered suggestive of obstruction (Carter et al, 1989; Vazquez-Levin et al, 1994; Smith et al, 2008).

Figure 21–2 Transrectal ultrasound (sagittal image) demonstrating a dilated ejaculatory duct culminating in an ejaculatory duct cyst.

Seminal vesicle aspiration using a 20-gauge needle at the time of TRUS has been used to further increase the specificity of the diagnostic techniques. Significant quantities of sperm are not normally present in the seminal vesicles. Findings of three or more sperm per HPF in the seminal vesicle aspirate support the diagnosis of EDO (Jarow, 1994). Test accuracy is improved by performing aspiration within 24 hours of ejaculation (Jarow, 1996).

Seminovesiculography using transrectal injection of radioopaque contrast (50% renograffin) into the seminal vesicles under TRUS guidance with postinjection radiographs can provide excellent anatomic detail of the seminal vesicles and ejaculatory ducts. Seminal vesicle chromotubation is a variation of seminovesiculography using the injection of dilute indigo carmine or methylene blue (1:5 dilution with saline) into the seminal vesicles via TRUS guidance followed by cystoscopic inspection of the ejaculatory ducts in the prostatic urethra to confirm patency. The dynamic tests of chromotubation and seminal vesiculography offer higher specificity for detection of EDO than static TRUS imaging alone (Purohit et al, 2004). The newest adjunctive technique, ejaculatory duct manometry, involves hydraulic assessment of the ejaculatory ducts at the time of seminal vesicle chromotubation, noting that men with EDO have higher mean ejaculatory duct opening pressures, 116 cm H₂O versus 33 cm H₂O in fertile controls (Eisenberg, 2008). Despite these advances in diagnostic techniques, criteria for diagnosis of complete EDO remain unclear and those for partial EDO remain controversial.

Scrotal Ultrasonography

Scrotal ultrasound for the infertile male is primarily used to confirm the presence of clinical varicoceles, although it also provides high-quality imaging of scrotal contents that offers the advantage of widespread availability without exposure to ionizing radiation. Although clinical varicoceles do not require confirmation with ultrasound examination, color Doppler ultrasound may be required when the clinical examination is difficult due to body habitus or when the examination is equivocal. Demonstration of reversal of venous blood flow with the Valsalva maneuver or spermatic vein diameters of 3 mm or greater (Fig. 21–3A and B) support the diagnosis of varicocele (Meacham et al, 1994). Scrotal ultrasound is not recommended for screening for subclinical varicoceles because repair of these has not been demonstrated to be of clinical benefit.

Figure 21–3 Scrotal ultrasound of varicocele. A, Dilated veins in spermatic cord. B, Color Doppler image revealing typical venous reflux with Valsalva maneuver.

In addition, ultrasound examination provides excellent anatomic details of the epididymis and testis, potentially disclosing a number of conditions that may affect fertility. Epididymitis is associated with epididymal enlargement with diffuse hypoechogenicity and is often associated with a reactive hydrocele. Doppler ultrasound will often reveal increased vascularity in the involved epididymis or adjacent testicular region. Epididymal cysts or spermatoceles appear as simple or minimally complex cysts and may cause epididymal outflow obstruction. Testicular germ cell tumors are noted with increased frequency in the subfertile population, and even small, nonpalpable lesions (<0.5 cm) are well visualized with ultrasonography (Fig. 21–4). Testicular microlithiasis are diffuse 1- to 2-mm hyperechogenic nonshadowing foci and have been reported in 3% of subfertile men (Thomas et al, 2000). Although originally thought to represent a radiologic precursor to the development of germ cell tumors, it is now known that testicular microlithiasis is common and does not represent a risk factor for germ cell tumor development or necessitate continued medical surveillance (Costabile and Spevak, 1998).

Figure 21–4 Testicular ultrasound—small nonpalpable testicular mass (seminoma) noted on fertility evaluation.

Abdominal Ultrasonography

Abdominal ultrasound imaging in the infertile male is primarily indicated to rule out associated renal anomalies in patients with vasal agenesis. Vasal agenesis is theorized to result from one of two mechanisms—either mutations in the CFTR gene that do not incur associated renal anomalies or improper morphogenesis of the mesonephric duct before week 7 of gestation, which causes vasal agenesis and unilateral renal agenesis.

The CFTR gene is responsible for regulation of chloride ion transport across epithelial cell membranes. Dysfunction in this transport results in abnormal luminal fluid quality during vasal morphogenesis with resulting vasal agenesis (Oates and Amos, 1993). In patients with proven CFTR gene mutation in association with CBAVD, renal anomalies are unlikely and routine abdominal imaging is not indicated. However, up to 20% of men with CFTR mutation-negative vasal agenesis will have ipsilateral renal anomalies, most commonly renal agenesis, and should have routine abdominal ultrasonography as part of their evaluation.

Vasography

Vasography remains the gold standard test for assessing the patency of the male ductal system. Although procedures such as TRUS, seminal vesicle aspiration, and seminal vesiculography offer minimally invasive imaging to diagnose obstruction, properly performed vasography provides unequalled anatomic detail of the vas deferens, seminal vesicles, and ejaculatory ducts. Vasography is indicated for determination of the site of obstruction in the azoospermic patient with confirmed normal spermatogenesis on testis biopsy. On occasion, it may be used for the severely oligospermic patient in whom there is a high clinical suspicion of unilateral vasal obstruction from iatrogenic injury such as prior inguinal hernia repair (Matsuda, 2000). Vasography may also be used to rule out ejaculatory duct obstruction in the setting of ejaculatory pain. However, it should be emphasized that vasography is not necessary in oligospermic patients who do not have clinical evidence or history (i.e., inguinal surgery) that suggests unilateral obstruction.

Vasography is ideally performed at the time of anticipated reconstruction due to the potential to cause vasal scarring at the vasogram site, although this complication has not been observed in large series (Payne et al, 1985). For antegrade vasography, the straight portion of the vas deferens is isolated in the scrotal region, immediately adjacent to the convoluted vas to allow maximum length of vas deferens if a reconstruction such as a vasoepididymostomy is eventually required. Either a puncture or vasotomy technique may be used to inject contrast into the vas deferens. The puncture technique is preferred if possible because it avoids a full-thickness vasotomy, which necessitates subsequent microsurgical closure, although it is technically more difficult to enter the vasal lumen than with the vasotomy method. With the puncture procedure, a 30-gauge lymphangiogram needle is inserted directly into the proximal vas lumen and contrast is injected in an antegrade fashion (Fig. 21–5A). Alternatively, a microsurgical scalpel may be used to make a hemivasotomy incision through the anterior wall of the vas deferens to expose the vasal lumen and allow placement of a 25-gauge angiocatheter for contrast injection (Fig. 21–5B). This techique allows examination of the intravasal fluid for presence of sperm to confirm epididymal patency. If a hemivasotomy technique is used for the vasogram, the vas will require reconstruction at the end of the procedure with 9-0/10-0 nylon interrupted sutures using standard microsurgical technique. Once the vasal lumen has been intubated, 5 to 10 mL of full- or half-strength contrast (Renografin) is injected in an antegrade fashion. Retrograde injection is not recommended due to the potential for subsequent epididymal scarring or obstruction. Vasography images are obtained using either standard radiographs or fluoroscopy. Methylene blue or indigo carmine may be mixed with contrast (1:10 dilution) to guide the depth of transurethral resection if the vasogram is performed at the time of ejaculatory duct resection. On occasion, contrast will not flow and a 2-0 monofilament suture may be passed in an antegrade fashion to identify the level of obstruction.

Figure 21–5 Vasography—examples of technique and findings. A, Technique for antegrade injection of contrast into proximal vas deferens. B, Normal vasogram with clearly defined vas deferens (VD), seminal vesicles (SV), ejaculatory duct (ED), and contrast spilling into bladder. C, Obstruction of left inguinal vas deferens due to prior herniorrhaphy. D, Ejaculatory duct obstruction with large cyst (arrow).

A properly performed vasogram will opacify the scrotal and inguinal portions of the vas deferens with subsequent filling of the ipsilateral seminal vesicle and the bladder (Fig. 21–5C). Failure to opacify the bladder represents either insufficient injection of contrast or evidence of obstruction (Fig. 21–5D).

Venography

Venography of the internal spermatic veins has been used to diagnose and treat varicoceles. As a diagnostic test, venography is arguably the most sensitive imaging modality but specificity remains its limitation. Although nearly 100% of clinical varicocele patients will demonstrate reflux on venographic examination, left internal spermatic vein reflux has been reported in up to 70% of patients without a palpable varicocele (Ahlberg et al, 1966; Narayan et al, 1980). False-positive studies may be due to examination technique factors such as high-pressure contrast instillation or placement of the catheter tip through a valve in the proximal portion of the internal spermatic vein. Because of the high false-positive rate and the invasive nature of the test, venography is not indicated for routine screening in the subfertile male. It does have utility in patients with presumed postvaricocelectomy recurrence both for confirmation of the diagnosis and embolization of persistent vessels (Fig. 21–6A and B).

Figure 21–6 Venography for evaluation and treatment of varicoceles. A, Venogram demonstrates reflux through incompetent venous valves of the left internal spermatic vein consistent with left varicocele. Inset reveals inguinal plexis of gonadal veins. B, Venous embolization procedure with catheter tip (white arrow) and deployed coils (black arrow).

Percutaneous embolization of varicoceles has been described using deployed coils, balloons, and sclerotherapy. Although it may be used for initial treatment, higher recurrence rates than surgical repair and unsuccessful procedure rates support embolization as a second-line therapy (Khera and Lipshultz, 2008). In addition, rare safety issues such as balloon migration, femoral vein injury, and anaphylactic reactions to contrast material further reinforce the recommendation that embolization is not the initial procedure of choice (Matthews et al, 1992; Zini et al, 1997). However, embolization does have a role in the management of patients with a persistent or recurrent varicocele after varicocele ligation. Antegrade scrotal sclerotherapy has also been described as first-line varicocele therapy with success rates as high as 95%, although this technique awaits validation with larger series and with long-term follow-up (Tauber and Johnson, 1994; Ficarra et al, 2002).

Normal

More than 85% of testicular volume consists of seminiferous tubules made up of progressively maturing germ cells and their supporting Sertoli cells. Blood vessels and Leydig cells in the interstitial areas provide the rest of the cellular complement. Spermatogenesis proceeds in an orderly fashion from spermatogonia along the basement membrane to spermatocytes and finally mature spermatozoa adjacent to the tubular lumen (Fig. 21–7A). Type A spermatogonia provide the stem cell complement and have a diploid complement of chromosomes (2N). These cells undergo a mitotic division to replenish the stem cell complement and provide type B spermatogonia (2N). Type B spermatogonia undergo DNA synthesis, resulting in spermatocytes that have a tetraploid complement of chromosomes (4N). Two subsequent meiotic divisions result in spermatids with the final haploid number of chromosomes (1N). Further maturation results in a progression from round to elongated spermatids and then to the final product, mature spermatozoa. Human testes exhibit multiple stages of spermatogenesis in a single tubular section (patchwork pattern), unlike other mammalian testes, which produce waves of spermatogenesis progressing along a tubule.

Figure 21–7 Diagnostic findings on testicular biopsy. A, Normal. B, Hypospermatogenesis. C, Maturation arrest. D, Germinal cell aplasia (Sertoli cell–only pattern).

In the setting of azoospermia, a normal testicular biopsy is considered pathognomonic of ductal obstruction. Although focal areas of hypospermatogenesis and maturation arrest have been observed in the presence of obstruction, particularly in the setting of prior vasectomy, these are not classic findings for obstruction (Joshi et al, 1972; Perera, 1978). Dilated tubules with intraluminal debris are also commonly noted in patients exhibiting obstruction.

Hypospermatogenesis

Hypospermatogenesis is associated with reduced numbers of all germ cells, but all stages of spermatogenesis remain present in the histologic section (Fig. 21–7B). The degree of reduction determines whether the patient is oligospermic or azoospermic. A critical level of sperm production is required before sperm can be detected in the ejaculate, accounting for the common observation of hypospermatogenesis in the azoospermic patient.

Maturation Arrest

As the name suggests, maturation arrest involves a block of sperm maturation at a specific stage anywhere along the path of spermatogenesis (Fig. 21–7C). Maturation arrest most commonly occurs at the primary spermatocyte or late spermatid stages. Late maturation arrest may be difficult to distinguish from a normal biopsy, but the presence of mature sperm on testicular touch prep supports the diagnosis of normal spermatogenesis. Complete maturation arrest will produce azoospermia, whereas patients with partial maturation arrest may present with severe oligospermia. The remaining testicular architecture including the Sertoli and Leydig cells, as well as the basement membranes, are usually normal. Interestingly, maturation arrest is often associated with normal endocrine profiles such as FSH and inhibin levels due to an intact hypothalamic-pituitary-gonadal negative feedback system. Thus the clinical presentation of maturation arrest and ductal obstruction may be similar because both are associated with normal testicular volume and similar endocrine profiles.

Germinal Aplasia

Germinal aplasia, also called Sertoli cell-only syndrome, has small seminiferous tubules that are completely devoid of germ cells (Fig. 21–7D). The interstitial component, as well as the Sertoli cells and basement membranes, are normal. The diagnosis of germinal aplasia is suggested in an azoospermic patient with small-volume testes and elevated levels of FSH consistent with primary testicular failure. Although there are no current treatments to repopulate the testis with germ cells, low levels of spermatogenesis may be seen in other areas of the testis, which forms the basis for the use of microsurgical dissection testis biopsy (micro-TESE) to retrieve sperm in some of these patients for eventual ICSI use.

End-Stage Testes

Thickened basement membranes, tubular and peritubular sclerosis, and absence of both germ cells and Sertoli cells are characteristic histologic findings for end-stage testes. These patients will have azoospermia with profoundly small, soft testes (2- to 3-mL volume). Although this is characteristic of KS, some patients may still have small foci of retained spermatogenesis, which may be identified by micro-TESE. This histology may also be seen with cryptorchid testes.

Azoospermia

Azoospermia is defined as the absence of sperm in the ejaculate and is identified in 10% to 15% of infertile males (Jarow et al, 1989). Before proceeding with further diagnostic procedures, the diagnosis of azoospermia should be confirmed with at least two centrifuged semen specimens to rule out severe oligospermia. The finding of even small quantities of sperm in the centrifuged specimen rules out complete ductal obstruction such as CBAVD and also offers the potential for immediate sperm cryopreservation for ICSI cycles.

Although there is a myriad of causes of azoospermia, etiologies fall into three general categories: pretesticular, testicular, and post-testicular. Causes can usually be differentiated on the basis of semen volume (low vs. normal), physical exam findings of testicular volume and presence of vas deferens as well as serum FSH level (Fig. 21–8).

Figure 21–8 Algorithm for evaluation and treatment of azoospermia. AID, artificial insemination by donor semen; CFTR, cystic fibrosis transmembrane conductance regulator; CT/MRI, computed tomography/magnetic resonance imaging; FSH, follicle-stimulating hormone; IVF, in-vitro fertilization; LH, leuteinizing hormone; PESA, percutaneous epididymal sperm aspiration; TESE, testicular sperm extraction; TRUS, transrectal ultrasound; TUREJD, transurethral resection of the ejaculatory ducts.

Pretesticular causes, also called secondary testicular failure, are usually endocrine in nature and relate to either congenital (Kallman syndrome) or acquired hypogonadotropic hypogonadism, which are addressed in detail later in this chapter.

Testicular etiologies, broadly termed as primary testicular failure, are intrinsic disorders of spermatogenesis. Direct testicular pathology may derive from genetic abnormalities such as Y-chromosome microdeletions or chromosomal abnormalities, varicocele-induced testicular damage, gonadotoxic effects from medications or environmental exposures, and idiopathic infertility, which constitute the majority.

Ejaculatory dysfunction or obstruction of the genital tract account for the post-testicular pathologies, which constitute 40% of cases of azoospermia (Best Practice Committees of American Urological Association and American Society for Reproductive Medicine, 2006). Pretesticular and post-testicular causes are often amenable to treatment, which may restore fertility, whereas the success rates for intervention in testicular pathology are much more modest.

Although both primary and secondary testicular failure will be associated with marked reduction in testicular volume, these entities can be distinguished by serum endocrine testing to include FSH, LH, testosterone, and prolactin levels. High serum FSH levels, typically greater than two times normal, are indicative of primary testicular failure, and diagnostic testicular biopsy is not required to rule out obstructive etiologies. Primary testicular failure in conjunction with azoospermia, commonly termed nonobstructive azoospermia (NOA), is best managed with testicular sperm harvest for eventual ICSI. The absolute degree of serum FSH elevation has not proven predictive of success rates for sperm retrieval (Vernaeve et al, 2004; Hibi et al, 2005; Harris and Sandlow, 2008). Azoospermic patients with normal testicular size, palpable vas deferens, and normal serum FSH levels require a diagnostic testicular biopsy to differentiate genital tract obstruction from disorders of spermatogenesis such as maturation arrest.

Obstructive azoospermia accounts for 40% of cases of azoospermia (Practice Committee, American Society of Reproductive Medicine, 2008). A normal testicular biopsy is pathognomonic for genital tract obstruction. If these patients desire restoration of patency, they will require scrotal exploration and vasography at the time of planned reconstruction to identify the site of obstruction. Genital tract obstruction may occur anywhere along the sperm transport system to include the rete testis, efferent ductules, epididymis, vas deferens, or ejaculatory ducts. Obstruction at the level of the epididymis or vas deferens may be successfully treated with microsurgical reconstruction, either vasoepididymostomy for epididymal obstruction or vasovasostomy for vasal obstruction. Elective vasectomy remains the leading cause of obstructive azoospermia and subsequent infertility. In the absence of prior injury to the vas either intentionally from vasectomy or iatrogenic injury of inguinal vas from hernia repair, most men with idiopathic ductal obstruction will be obstructed at the level of the epididymis. Epididymal obstruction may be due to prior trauma, infection, or the result of downstream vasal obstruction from an elective vasectomy. Although vasectomy usually results in a single obstructive site, the development of high intraluminal pressures after a vasectomy can result in rupture of the delicate epididymal tubule with secondary obstruction in the epididymis. Epididymal obstruction rarely occurs within 4 years of a vasectomy but is present in more than 60% of patients on one or both sides after 15 years of vasal obstruction (Fuchs and Burt, 2002). The absence of proximal vasal fluid or thick, viscous, pastelike fluid is indicative of a concurrent epididymal obstruction and such patients will require a vasoepididymostomy for their reconstruction. The outcome of reconstruction in vasectomized men depends on a number of factors including the obstructive interval, the quality of the fluid from the proximal vas at the time of surgery, and the surgeon’s microsurgical experience (Sabanegh, 2009).

Azoospermia in conjunction with low semen volumes and palpable vas deferens is most likely caused by ejaculatory duct obstruction (EDO), although rarely, ejaculatory dysfunction can cause a similar presentation. Disorders of ejaculation such as retrograde ejaculation will more frequently cause oligospermia in conjunction with low semen volume and are readily diagnosed by the presence of sperm in a postejaculation urine specimen. The diagnosis of EDO is suggested by low semen volume with acidic pH and absent semen fructose. TRUS will often reveal midline prostatic cysts, dilated ejaculatory ducts, or dilated seminal vesicles (>1.5 cm in width), and seminal vesicle aspiration may produce large numbers of sperm. Ultimately, EDO may be confirmed by vasography at the time of planned transurethral resection of the ejaculatory ducts (TUREJD) to relieve the obstruction.

Oligospermia

Oligospermia is defined as a sperm density of less than 20 million/mL. Unlike the situation with azoospermia, the number of potential diagnoses causing oligospermia can be quite vast and the etiology is often idiopathic. Oligospermia is rarely seen as an isolated seminal abnormality but is usually associated with disturbances in motility and morphology. Endocrinopathies are rarely observed in patients with concentrations higher than 10 million/mL, so routine endocrine screening with serum testosterone and FSH levels are reserved for counts lower than 10 million (Sigman and Jarow, 1997). Testis biopsy is not indicated in the setting of mild to moderate oligospermia, although it can be considered in patients with sperm concentrations under 1 million/mL in whom ductal obstruction is considered on the basis of history or physical examination findings.

Seminal Quality Abnormalities

Isolated defects may occur in motility or morphology, known as asthenospermia or teratospermia, respectively, while global sperm quality abnormalities are described with the term oligoasthenoteratospermia (OAT). Asthenospermia may be iatrogenic from delayed processing in the laboratory or may be due to a prolonged abstinence period. Persistent asthenospermia in a properly processed specimen, while often idiopathic, may be seen in association with varicoceles, genital tract infections, ultrastructural cilia abnormalities such as immotile cilia syndrome, and immunologic infertility in association with antisperm antibodies.

Teratospermia is a common finding, especially with use of the strict morphologic criteria (Tygerberg or Kruger) applied by many andrology laboratories. Bizarre morphologies of the sperm head such as pinhead sperm, multiheaded sperm, or round-headed sperm, which suggest acrosome deficiency, undoubtedly have clinical significance, although there is growing controversy regarding the predictive utility of abnormal morphology in general, largely due to the subjective nature of the test making it difficult to standardize (Agarwal et al, 2008). Abnormal sperm morphology has not been correlated with recurrent spontaneous miscarriages or abnormalities in offspring (Rosenbusch et al, 1992; Hill et al, 1994).

Low ejaculate volume may also be a contributing factor to subfertility. Reduction in ejaculate volume is most commonly due to improper specimen collection but may be associated with CBAVD with associated hypoplasia of the seminal vesicles, hypoandrogenism, retrograde ejaculation, or obstruction of the ejaculatory duct. Relative acidity of the seminal pH and low semen fructose suggest absence of seminal vesicle contribution to the semen, pointing to either CBAVD or EDO as lead possible causes of subfertility.

Normal Bulk Semen Parameters

Up to 50% of men presenting for an infertility evaluation will have normal bulk semen parameters, representing a particularly difficult population to be assigned an etiology for subfertility and reinforcing the inherent inability of standard semen analyses to assess sperm function. In these couples, particular attention should be given to identifying occult female factor fertility issues, as well as assessing coital frequency to optimize reproductive timing for conception. On occasion, antisperm antibodies in the cervical mucus may inhibit sperm motility in vivo and prevent fertilization. This situation can be quantified with indirect antisperm antibody testing of the cervical mucus, although an in-vivo assessment of compatibility of sperm with the cervical mucus can be provided with the postcoital test (PCT). Couples with an abnormal PCT may benefit from intrauterine insemination, which bypasses the hostile cervical factors.

Hypogonadotropic Hypogonadism

Isolated gonadotropin deficiency or hypogonadotropic hypogonadism (HH) is a relatively rare cause of subfertility accounting for less than 1% of cases of male infertility and may be congenital or acquired (Sigman and Jarow, 1997).

Acquired causes of hypogonadotropic hypogonadism include pituitary disease, which may result from prior surgery, infarction, tumors, or infection; metabolic disorders; and a variety of other medical conditions (Table 21–12).

Table 21–12 Acquired Causes of Hypogonadotropic Hypogonadism

Although variants of congenital hypogonadotropic hypogonadism have been described (Table 21–13), the anosmic form or Kallman syndrome is the most commonly reported, occurring in between 1:10,000 and 1:60,000 births (Oates, 1997). In addition to the anosmia and azoospermia noted with Kallman syndrome, other clinical features may include midline facial defects such as cleft palate; gynecomastia; neurologic abnormalities (mental retardation, oculomotor defects, deafness, and synkinesia); unilateral renal agenesis; cryptorchidism; micropenis; and pes cavus (Sussman et al, 2008). Kallman syndrome results from failure to secrete gonadotropin-releasing hormone (GnRH) by the hypothalamus, leading to low gonadotropin levels and ultimately, failure to transition the prepubertal testis to postpubertal level of function. The GnRH deficiency noted with Kallman syndrome is the result of failed embryonic migration of neuroendocrine GnRH cells from the olfactory epithelium to the forebrain. Absence or hypoplasia of the olfactory bulbs accounts for the concurrent anosmia. Kallman syndrome represents a genetically diverse group of diseases with both X-chromosome linked inheritance via mutations in the KAL1 gene, as well as autosomal dominant inheritance via mutations in the fibroblast growth factor receptors 1 and 8 (FGFR1, FGFR8), the prokineticin receptor-2 (PROKR2), and the prokineticin-2 (PROK2) genes. Mutations in these five genes are found in less than 30% of Kallman syndrome patients reinforcing the need for further genetic study on these complex patients (Dode, 2009).

Table 21–13 Variants of Congenital Hypogonadotropic Hypogonadism

Prader-Willi syndrome (PWS), another form of congenital hypogonadotropic hypogonadism, has features that include infantile hypotonia, obesity, cryptorchidism, short stature, and mental retardation. It is caused by microdeletions or mutations on the paternal chromosome 15 at the q11 or q13 location (Smeets et al, 1992). Because of concurrent medical problems in these patients, most do not seek treatment for infertility.

Hypogonadotropic hypogonadism usually presents in the late teenage years when the patient fails to undergo the usual pubertal changes with secondary virilization. It is of critical importance to distinguish HH from constitutional delay of growth and puberty (CGDP) because they share many clinical and hormonal features and yet require markedly different treatments. Usually, CGDP patients will attain spontaneous puberty by age 18 or may require transient androgen replacement for puberty induction but then will progress through normal development and fertility. HH patients will require lifelong androgen replacement for maintenance of virilization and episodes of exogenous gonadotropin treatment off androgen therapy for fertility induction as described later. Because differentiation is not always possible on the basis of serum testosterone and gonadotropin levels, a number of physiologic and stimulatory tests have been described to discriminate these conditions to include serial serum LH measurements, prolactin response to TRH, and GnRH and human chorionic gonadotropins (HCG) stimulation tests (Segal, 2009). Patients with CGDP often demonstrate LH pulses with serial overnight blood draws and gonadotropin surge in response to GnRH stimulation. HH patients have minimal variability in LH levels and do not demonstrate significant increases in gonadotropins levels with administration of GnRH.

In general, treatment of HH syndromes is directed to two ultimate goals: (1) induction of normal serum androgen levels to allow appropriate virilization and bone growth and (2) the induction of spermatogenesis and, ultimately, fertility. During adolescence, puberty and virilization are usually initiated with exogenous androgen treatment. Various effective methods of testosterone replacement therapy exist including intramuscular testosterone enanthate or cypionate (200 mg every 2 weeks), transdermal patches (5 to 10 mg/day), testosterone gel, or buccal tablets. Adequacy of replacement can be assessed by serum testosterone levels, which should be within the midrange of normal, and observation of the desired phenotypic response to therapy. Exogenous testosterone is effective in induction of virilization but will inhibit spermatogenesis, so alternate therapies will be required when the patient desires fertility.

Induction of spermatogenesis in the HH patient requires the maintenance of intratesticular testosterone levels that are exponentially higher than that obtainable with conventional androgen replacement therapy. To obtain such levels, the usual first-line therapy for HH involves treatment with human chorionic gonadotropin (hCG), which is biologically similar to LH, inducing Leydig cell production of testosterone. hCG is administered subcutaneously at a dose of 1500 to 2000 IU two to three times per week for 4 to 6 months. When testicular volume and serum testosterone levels are stable without significant further improvement, FSH stimulation is added to the treatment regimen to induce spermatogenesis. FSH may be administered in the form of human menopausal gonadotropin (hMG), a compound that contains both FSH and LH in equal doses or with recombinant human FSH formulations. Human menopausal gonadotropin is administered at a dose of 75 IU two to three times per week, and recombinant FSH usually uses doses of 37.5 to 75 IU three times per week. FSH therapy is continued until attainment of sperm concentrations of at least 5 million per mL in the ejaculate or pregnancy is obtained. The vast majority of patients will be able to conceive after gonadotropin therapy, although 71% of the patients with subsequent fertility have sperm concentrations considerably lower than normal, suggesting that these patients are often able to conceive with low counts (Burris et al, 1988). Interestingly, 10% of patients may maintain normal serum testosterone levels after cessation of all endocrine therapy, implying that HH may be reversible in some patients (Raivio et al, 2007). Prior treatment of HH patients with testosterone does not affect success with future gonadotropin therapy (Ley and Leonard, 1985; Hamman and Berg, 1990).

Hypogonadotropic hypogonadism patients with intact pituitary function can also be effectively treated with pulsatile GnRH therapy supplied via subcutaneous infusion from portable pump or intranasal administration (Klingmuller, 1985; Aulitzky, 1988). Although direct GnRH therapy is effective, inconvenience of administration and expense do not justify this choice over traditional gonadotropins therapy at the present time (Liu, 1988). In addition, selected patients with HH presenting after puberty with intact pituitary function may respond to clomiphene citrate, an antiestrogen therapy (Whitten et al, 2006).

Androgen Excess

Excess systemic levels of androgen paradoxically inhibit spermatogenesis due to direct feedback inhibition of gonadotropin secretion at the level of the hypothalamus and pituitary. This ultimately results in low intratesticular levels of testosterone, which are inadequate for the maintenance of spermatogenesis, an observation that provides the impetus for growing research examining the potential contraceptive role of exogenous testosterone.

Excess states may result from either endogenous production or exogenous administration of androgens. Elevations in endogenous production of androgens may be caused by congenital abnormalities of steroid metabolism such as congenital adrenal hyperplasia or androgen-producing tumors of the testis or adrenal gland. Congenital adrenal hyperplasia (CAH), the most common endogenous etiology of androgen excess, encompasses a variety of specific enzyme defects in cortisol and aldosterone synthesis. Inadequate systemic cortisol levels result in excessive pituitary release of ACTH leading to hyperstimulation of the adrenal gland with subsequent release of adrenal androgens and suppression of pituitary gonadotropin release. Deficiency in the 21-hydroxylase enzyme accounts for 90% of cases of CAH. Although severe deficiency in 21-hydroxylase activity can present in the neonatal period with salt wasting, milder deficiencies present more commonly in childhood with phallic enlargement, precocious puberty, and advanced skeletal maturation. Elevated levels of serum 17-hydroxyprogesterone (often >1000 ng/dL) and urinary pregnanetriol support the diagnosis of 21-hydroxylase deficiency. The impact on subsequent fertility is variable, depending on the degree of androgen excess. Treatment for CAH consists of glucocorticoid replacement, which reduces ACTH levels and adrenal androgen production. In one small series of adult patients with CAH, 60% of men were able to cause pregnancies either with treatment or even in the absence of CAH treatment (Urban, 1978).

Anabolic steroids represent a growing etiology of male subfertility with a similar mechanism of spermatogenic disruption to that observed with CAH. It remains problematic to accurately predict the impact of these agents because of the variety of performance-enhancing substances used, as well as the variation in doses employed. Older studies of semen quality in athletes using high doses of anabolic steroids revealed severe impairment of sperm concentration, motility, and morphology (Knuth et al, 1989). Although cessation of use allowed normalization of seminal parameters in an average of 4 months, cases of persistent azoospermia have been reported occurring more than 1 year later (Jarow, 1990). To avoid the fertility-related side effects, anabolic steroid users have employed modified regimens that add hCG with steroid to protect spermatogenesis. These combinations, although causing transient impairment in semen quality, seem to allow preservation of spermatogenesis in some patients (Karila et al, 2004). In patients with persistent azoospermia or severe oligospermia after steroid abuse, hCG alone or in combination with hMG has been successfully used to restore spermatogenesis (Pundir, 2008).

Hyperprolactinemia

Because of its low yield, screening serum prolactin measurements in the infertile male is not indicated unless associated with erectile dysfunction, low serum testosterone levels, or decreased libido. Although often idiopathic, hyperprolactinemia can be associated with medications, physiologic stress, and pituitary tumors. Because of the marked physiologic variability noted with serum prolactin levels, elevated levels should be confirmed by at least one additional measurement. If elevated prolactin levels are confirmed (>18 ng/dL), anatomic imaging of the sella turcica is required, usually with magnetic resonance imaging with gadolinium contrast. Although macroadenomas may require surgical excision, microadenomas are usually responsive to dopamine agonist therapy with either bromocriptine or cabergoline. Cabergoline offers advantages over bromocriptine to include longer half-life, allowing less frequent dosing and an improved side effect profile without compromising therapeutic efficacy. Despite limited data on the therapeutic efficacy of dopamine agonist therapy for the infertile male with hyperprolactinemia, several small studies have reported improvements in sperm concentration and motility, as well as normalization of serum prolactin levels with treatment (Laufer et al, 1981; Mancini et al, 1984).

Estrogen Excess

Regardless of the source of the estrogen excess, estrogens inhibit GnRH secretion by the hypothalamus and gonadotropin release by the pituitary. In addition to the effects on the hypothalamic-pituitary-gonadal axis, elevations in serum estrogen levels produce direct deleterious effects on spermatogenesis (Hammoud et al, 2008). Hormonal evaluation in the infertile patient will reveal low levels of serum FSH, LH, and testosterone with elevations in serum estrone and estradiol (E₂).

In the male, hyperestrogenemia may result from hepatic disease, estrogen-producing tumors, or, most commonly, obesity. Sertoli or Leydig cell testicular tumors and adrenal cortical tumors may rarely produce elevated estrogen levels. In the obese patient, elevations in serum estrogen derive from elevated peripheral aromatization of androgen to estrogen occurring in adipose tissue via an aromatase enzyme. The level of serum androgen inversely correlates with the degree of obesity (Giagulli et al, 1994; Tchernof et al, 1995). This provides the scientific basis for the use of selective medical therapy with aromatase inhibitors in men with reduced testosterone-to-estrogen ratios, which are discussed later in this chapter. In the obese patient, weight loss and bariatric surgery have been associated with improvements in endocrine profiles (Kaukua et al, 2003; Globerman et al, 2005), although the ultimate effect on fertility remains subject to further study.

Disorders of Sex Chromosome Number and Structure

Y-Linked Microdeletions

Testing for Y chromosome microdeletion is indicated in men with presumed nonobstructive etiology of azoospermia and severe oligospermia (concentration < 5 million/mL) to assess the prognosis for surgical sperm retrieval and to assist genetic counseling for affected couples. Normal molecular anatomy of the Y chromosome is critical both for gonadal development and function. The Y chromosome is an acrocentric chromosome with an off-center centromere creating asymmetric short (Yp) and long (Yq) arms incorporating 60 million base pairs (Morton, 1991). The Y chromosome has two important regions—the euchromatic zone, which includes Yp, the centromere, and the proximal portion of Yq, and the heterochromatic zone, which comprises the distal Yq. The heterochromatic region does not appear to have transcriptional function, while the euchromatic zone has a number of important genetic loci that are critical to the development of normal spermatogenesis (Fig. 21–9). Of particular importance is the sex-determining region Y (SRY) gene located on Yp, which is critical in initiating the cascade that converts the embryonic bipotential gonad to the eventual testis.

Figure 21–9 Y chromosome with azoospermia factor (AZF) region.

Deletions or mutations of critical areas of the Y chromosome may cause profound disruption of spermatogenesis. Early cytogenetic analysis of infertile men suggested the presence of a specific region on Yp termed the azoospermia factor (AZF), which is of paramount importance for normal spermatogenesis (Tiepolo and Zuffardi, 1976). Subsequent advances in chromosomal molecular mapping allowed the identification of three regions of Y-chromosome microdeletion: AZFa, AZFb, and AZFc (Vogt, 1998). Deletions in the AZFa region have been reported in 1% of men with nonobstructive azoospermia. This region contains two genes, DDX3Y (also known as DBY) and USP9Y, which are felt to be important for normal spermatogenesis (Kamp et al, 2000). AZFa microdeletion is associated with germinal cell aplasia histology of the testis, and current literature suggests that an attempt at testicular sperm retrieval (TESE) is not indicated because the success rate is poor (Blagosklonova et al, 2000; Hopps et al, 2003; Oates, 2008).

Like AZFa, microdeletions in the AZFb region are uncommon and are associated with poor surgical sperm retrieval success rates, especially if associated with concurrent microdeletion in other loci. The critical spermatogenic gene in the AZFb region is the RNA-binding motif (RBM) gene, which produces an RNA-binding protein localized to germ cell nuclei (Ma et al, 1993). Microdeletion in the AZFc region is the most most commonly noted, in up to 13% and 6% of azoospermic and severely oligospermic men, respectively (Reijo et al, 1996). The AZFc region is the location for the Deleted in Azoospermia (DAZ) gene, which encodes an RNA-binding protein noted primarily in spermatogonia (Saxena et al, 2000; Collier et al, 2005). Isolated AZFc microdeletion portends a better prognosis because more than 50% of azoospermic men will have successful sperm retrieval (Silber et al, 1998; Oates 2002).

Considering the fact that Y chromosome microdeletions do not have direct health implications to patients other than in the fertility arena, couples should receive genetic counseling before obtaining and using surgically retrieved sperm for ICSI. The male offspring of these patients will be phenotypically normal but will harbor the father’s microdeletion and thus are expected to have similar fertility challenges in their adulthood (Silber and Repping, 2002).

Disorders of Internal Ductal Development

Ultrastructural Sperm Anomalies

Functional Ejaculatory Dysfunction

Functional disorders include premature and delayed ejaculation. Premature ejaculation (PE) is a common form of male sexual dysfunction, occurring in more than 30% of men between the ages of 18 and 60 (Laumann et al, 1999). PE may cause significant emotional distress in couples, yet the vast majority of patients will still have intravaginal ejaculation minimizing the infertility implications of this condition. If ejaculation is consistently occurring before intromission, semen may be collected by the couple and used for intravaginal insemination at home.

Delayed ejaculation is defined as persistent difficulty or inability to ejaculate despite the presence of sexual desire and erection. No clear neurologic basis for this condition has yet been established and it is widely believed to represent a psychogenic issue (Ohl et al, 2008). Some authors have described an association with strict religious upbringing and frequent use of masturbation (Stewart and Ohl, 1989; Perelman, 2006). Some men may benefit from sex therapy or vibratory stimulation to induce ejaculation, but many will require electroejaculation or surgical sperm retrieval for eventual fertility.

Neurogenic Anejaculation

Neurogenic anejaculation is usually the result of a spinal cord injury (SCI). In addition to the ejaculatory dysfunction in these patients, many will also have erectile dysfunction and deficiencies in spermatogenesis related to thermal dysregulation of the testes, stasis of spermatozoa, and chronic genital tract infections. Oral therapies are rarely of value in these patients and most require penile vibratory stimulation (PVS), electroejaculation (EEJ), or surgical sperm retrieval to circumvent the lack of ejaculation.

Penile vibratory stimulation involves direct vibratory stimulation of the penile frenular region using specially designed equipment. Specific vibratory parameters including a vibration amplitude of 2.5 at 100 Hz appear optimal for ejaculation induction in SCI men (Sønksen, 1994). The best candidates for PVS are patients with complete SCI who have an intact ejaculatory reflex system: Specifically, patients with complete upper motor neuron lesions above T10 with preservation of sacral efferents, thoracolumbar sympathetic outflow, and intact communication between sacral and thoracolumbar segments will exhibit the best response to PVS because approximately 70% of these patients will have ejaculation in response to treatment (Ohl et al, 1996; Bird et al, 2001). Men with incomplete spinal cord lesions may have poor response to PVS because of cortical inhibition of the reflex arc preventing ejaculation. In addition, patients with lower spinal cord lesions or peripheral neural root injuries are unlikely to respond to PVS. The most serious adverse event associated with both PVS and EEJ is autonomic dysreflexia, a massive sympathetic discharge generally associated with SCI above the T6 level. Blood pressure monitoring is essential during any procedure on SCI patients. Men who are prone to autonomic dysreflexia may be given sublingual nifedipine, 10 to 20 mg 10 minutes before the procedure to minimize the blood pressure elevation (Steinberger et al, 1990). In general, PVS is well tolerated and can even be performed by the patient at home in the absence of autonomic dysreflexia. Sønksen reviewed the efficacy of PVS in SCI men reporting 102 pregnancies in 619 couples using home vaginal insemination or clinic intrauterine insemination (Sønksen and Biering-Sørenson, 1992). PVS remains the first-line therapy for SCI patients, producing better ejaculate quality than noted with EEJ procedures (Brackett et al, 1997; Ohl et al, 1997).

Electroejaculation may be used for SCI men who have failed a trial of PVS and is usually effective for any level of spinal cord injury, as well as in men with anejaculation from a variety of other mechanisms including retroperitoneal nerve injury from prior surgery such as retroperitoneal lymphadenectomy, diabetic neuropathy, multiple sclerosis, spina bifida, and psychogenic anejaculation. In the EEJ procedure, a rectal probe is inserted and pulsed direct electrical stimulation is applied to induce ejaculation. Although SCI patients often do not require an anesthetic during the procedure, men who retain normal pelvic and perirectal sensation will need general or spinal anesthesia. Because retrograde ejaculation often results, patients should have bladder catheterization before the procedure to minimize urine contact with the ejaculate. In addition, instillation of a sperm-friendly buffered medium into the bladder, systemic alkalinization with sodium bicarbonate, and hydration before the procedure will improve sperm survival in the bladder. Antegrade ejaculate is collected and the patient is again catheterized at the end of the procedure to recover any retrograde ejaculate. Sigmoidoscopy is also performed before and after the procedure to evaluate for preexisting rectal pathology that could interfere with the EEJ procedure, as well as identify any injuries resulting from the electrical stimulation of the rectal wall. Rectal injuries are rare, occurring in less than 0.1% of EEJ procedures, but they require prompt identification and treatment to minimize morbidity (Ohl and Sønksen, 1997). Electroejaculation procedures have been reported to produce ejaculates adequate for intrauterine insemination in 71% of men with SCI and 87% of patients with anejaculation postretroperitoneal lymph node resection (Ohl, 1989, 1995).

Regardless of the etiology of anejaculation, surgical sperm retrieval via either percutaneous or open biopsy of the epididymis or testis remains an effective option for management. One study conducted a cost-benefit analysis of EEJ coupled with intrauterine insemination versus ICSI using surgical sperm retrieval in SCI patients, noting that EEJ was the cost-effective approach if the procedure could be performed without anesthesia. However, in those patients requiring anesthesia for the EEJ, the cost-benefit analysis favored surgical sperm retrieval via local anesthetic followed by ICSI (Ohl, 2001).

Retrograde Ejaculation

Retrograde ejaculation occurs when seminal fluid preferentially flows into the bladder, instead of the normal antegrade direction due to failure of the bladder neck to close. The diagnosis is confirmed by identification of 10 to 15 sperm per HPF in a centrifuged specimen of postejaculation urine. The causes of retrograde ejaculation include incompetence of the bladder neck usually from prior surgery such as transurethral resection of the prostate gland, medications including α blockers and antidepressants, and diseases causing neurologic pathology such as diabetes and multiple sclerosis. Before initiating therapy, potential reversible etiologies such as medications should be removed if possible. In the absence of correctible etiologies, a trial of sympathomimetic medication therapy may be useful in increasing sympathetic tone of the bladder neck and vas deferens, especially in patients with slowly progressive disease such as diabetic neuropathy or in those with failure of emission due to disruption of retroperitoneal sympathetic innervation from prior surgery. Table 21–14 lists some of the standard medication regimens described. Medical therapy can produce a spectrum of results including conversion of retrograde to antegrade ejaculation, increase in ejaculatory volume, and development of retrograde ejaculation in a previously anejaculatory patient (Brooks et al, 1980; Kamischke and Nieschlag, 2002). These regimens must be used judiciously because side effects include tachycardia and hypertension, which may be particularly concerning in the diabetic patient at risk for occult cardiovascular disease.

Table 21–14 Medication Regimens for Treatment of Retrograde Ejaculation

Patients with retrograde ejaculation who do not respond to medical therapy may have sperm recovered from the bladder for use in intrauterine insemination as detailed earlier. With proper preparation of the bladder to retain sperm viability and laboratory sperm processing, this technique is an effective and economical method of treatment (Van der Linden, 1992).

Empiric Medical Therapy