Numerous changes in diagnostic and therapeutic decision-making strategies for the infertile male have been made in the last several years as a result of recent technological advances. One of the most important of these advances has been the introduction of the micromanipulation procedure known as intracytoplasmic sperm injection (ICSI). This technique has forever changed the practice of andrology by now enabling men who were previously thought to be irreversibly infertile the chance to initiate their own biologic pregnancy. As an example, the indications for obtaining a testis biopsy and interpretation of its results are quite different now that sperm can be harvested from a biopsy specimen and used with ICSI to initiate a fertilization. Another important technical advance has been seen with an increase in ultrasound utilization for visualizing the ductal system. Transrectal ultrasound-guided seminal vesiculography, rather than vasography, is now often the first line diagnostic modality for confirming ejaculatory duct obstruction. Using case presentations for clinical introductions, this syllabus highlights these important new developments.

The primary indication for vasography is the assessment of vasal obstruction within the inguinal vas deferens. As discussed previously, seminal vesicle aspiration and vesiculography have replaced standard vasography as the initial diagnostic method of choice for the assessment of ejaculatory duct obstruction. An inguinal vasal obstruction should be suspected in an azoospermic patient with normal spermatogenesis and a history of prior inguinal or scrotal surgery. In this situation, vasography should only be performed at the time of a potential microsurgical reconstruction.

Another situation in which we perform vasography is during a vasovasotomy or vasoepididymostomy. In these cases, we do not typically use contrast, but, rather, flush 10 cc of saline towards the prostate. If resistance is encountered, raising suspicions for an obstruction, then methylene blue may be added to the irrigating solution, or formal vasography may be performed. Alternatively, a 2-0 Prolene suture may be gently threaded within the vas deferens toward the prostate. The length of suture passed is used to estimate the site of obstruction.

Vasography may be performed using an open or puncture technique. Although there are numerous types of open techniques, we prefer making a hemivasotomy, or partial transection, at the junction of the straight and convoluted portions of the vas deferens. A 25 gauge, 1/2" Angiocath sheath is gently placed into the prostatic end of the vas deferens and a dilute nonionic contrast injected. One should never inject the contrast towards the epididymis because of the potential for damaging the delicate epididymal tubules. By tilting the table approximately 15° caudally (reverse Trendelenburg), the best quality radiographs may be obtained because the potential blocking effect of the symphysis pubis is avoided. For normal criteria, the reader is referred to a study by Banner. The presence of obstruction is confirmed by identifying the site of blockage, as well as evidencing resistance to the injection of the contrast. If methylene blue has been added to the nonionic contrast, a small urethral catheter may be placed into the bladder to assess for the blue discoloration if the diagnosis is still in doubt.

If an epididymal obstruction is suspected based on the finding of dilated epididymal tubules and the lack of distal obstruction, then an epididymovasostomy should be performed at the same operative setting. If an inguinal vas deferens obstruction is diagnosed, then one should proceed with a vasovasostomy. If an ejaculatory duct obstruction is diagnosed, then the hemivasotomy site should be closed with standard microsurgical technique. We use interrupted 10-0 nylon sutures for the inner layer, and interrupted 9-0 nylon sutures for the outer layer. We prefer the open method because in experienced hands the potential for submucosal injury to the vas deferens is minimized.

Alternatively, the puncture technique employs a 30 gauge lymphangiogram needle placed through the wall of the vas deferens in the direction of the prostate. Contrast or saline may then be injected distally. In less experienced hands, one risks creating submucosal false passages within the vas deferens if the lumen is not entered properly. The puncture technique and a partial thickness vasotomy have been compared in a rat model with the findings that fertility studies, vasal patency, sperm granuloma formation, and flow characteristics were roughly equivalent.

Because of its excellent visualization of the seminal vesicles, prostate, and ejaculatory ducts, TRUS has become an important diagnostic technique in the evaluation of the infertile male. TRUS for infertility purposes is commonly performed with the patient in the lateral decubitus, knee-to-chest position using a high-resolution, 6.5–7.5 MHz probe. Performance of the procedure with the bladder partially filled results in better imaging because of the acoustic window provided for delineation of the bladder, perivesical fat, and seminal vesicles. Indications and an in-depth discussion of techniques and findings for TRUS have been presented in-depth in the Journal of Clinical Ultrasound (vol. 24, p. 437, 1996).

A significant recent advance is the use of TRUS as the first-line diagnostic modality for evaluation of ejaculatory duct obstruction. Another significant purpose of TRUS is the assessment of the absence or hypoplasia of the seminal vesicles and ejaculatory ducts. Men with ejaculatory duct obstruction may present with primary infertility and azoospermia (no sperm present) or severe, unexplained oligoasthenospermia (severely depressed sperm density and motility). Thus, absolute indications for performing TRUS include low-volume azoospermia and low-volume, severe oligoasthenospermia in the absence of testicular atrophy and a normal serum concentration of FSH. Another absolute indication for performing TRUS is the presence of a midline cyst or asymmetry palpated on digital rectal examination. Certainly, one has first to be sure that retrograde ejaculation is not present. TRUS is also indicated in men with severe oligozoospermia and a low-volume ejaculate because a partial ejaculatory obstruction may be present. While a partial ejaculatory duct can cause a low volume severe oligozoospermia, a functional ampullo-vesicular seminal tract disorder must also be considered.

Ejaculatory duct cysts, ejaculatory duct calcification, ejaculatory duct dilation, and seminal vesicle dilation visualized on TRUS are all consistent with ejaculatory duct obstruction. Although obstruction should be suspected in patients with a transaxial seminal vesicle width of greater than 1.2–1.5 cm, seminal vesicle dilation does not occur in every patient, especially if a concurrent epididymal obstruction due to a "blowout" is present. Investigators have also looked at the endorectal coil MRI to provide a highly detailed anatomic depiction of the pelvis. However, because of lesser costs and wider availability, TRUS remains the procedure of choice for imaging the ejaculatory ducts and prostate for infertility related pathology.

The ejaculatory duct measures approximately 4–8 mm in diameter with a 2 mm lumen, and may be difficult to image in its typically nondilated state. The ejaculatory duct is formed by the confluence of the seminal vesicle and the terminal ampullary portion of the vas deferens. The ampulla of the vas deferens can be imaged in both the transverse and sagittal planes. They appear as a pair of oval, convoluted, tubular structures medial to the seminal vesicles and cephalad to the prostate. The obstructed lumen of the ejaculatory duct may be best appreciated in sagittal images as a hypoechoic tubular structure entering the urethra at the level of the verumontanum.

Intraprostatic cysts causing obstruction of the ejaculatory duct may be of Müllerian duct, Wolffian duct, or prostatic origin. Also known as utricular cysts, Müllerian duct cysts are midline in location and do not contain sperm. In a series of 150 consecutive infertility patients, Jarow demonstrated an 11% incidence of Müllerian duct cysts in contrast to 0% in the control group. These were the only cysts significantly associated with infertility and might have caused obstruction of the ejaculatory duct by external compression. Wolffian duct cysts, also known as ejaculatory duct diverticula, are also located in the midline. These cysts do contain sperm and may be produced by a distal obstruction of the ejaculatory duct. Prostatic retention cysts are located in the periphery of the prostate and do not contain sperm. Jarow found these cysts in 4 of the 150 infertility patients.

TRUS combined with seminal vesiculography has greatly reduced the need for the more invasive, open vasography, still considered the "gold standard" for identifying distal ejaculatory duct obstruction, but carrying a significant risk of vasal scarring.

With a 35 cm long, 21 gauge Williams needle (or a 30 cm, 17 gauge oocyte retrieval needle), the seminal vesicles may be aspirated transrectally under ultrasound guidance.

The presence of numerous sperm in the seminal vesicles is suggestive of obstruction at the ejaculatory duct in men with azoospermia or severe oligozoospermia. Because numerous motile sperm are not typically observed in the seminal vesicle aspirate from a normal patient, it is believed that in a complete ejaculatory duct obstruction, sperm can reflux into the seminal vesicles. A recent report suggests that aspiration should be performed immediately after ejaculation (within the same day), because small numbers of sperm may be found in the seminal vesicles in normal men with just 5 days of abstinence. This technique may be especially helpful in identifying the unilaterally obstructed ejaculatory duct when sperm are present in the ejaculate, but ultrasonographic findings are equivocal.

Seminal vesiculography may be performed after transrectal aspiration with the injection of non-ionic contrast. While this procedure may be performed without anesthesia, we find that mild sedation is helpful. Antimicrobial prophylaxis should be provided with a fluoroquinolone agent. Fluoroscopy, combined with radiographs after instillation of 5, 10, and 20 cc of contrast, provides ideal imaging. In certain cases, this technique can be used to document the patency of the pelvic and inguinal portions of the vas deferens.

During the injection of contrast with methylene blue, an assistant may view the verumontanum and ejaculatory ducts with a flexible cystoscope for further assessment of obstruction. If the methylene blue is visualized effluxing from an ejaculatory duct, a complete ejaculatory duct obstruction can be excluded. Digital massage of the seminal vesicles after removal of the TRUS probe assists the efflux of fluid. The failure of contrast of methylene blue to appear in the bladder confirms the diagnosis of an ejaculatory duct obstruction.

CASE PRESENTATION:

1. Semen analyses (x 2): Volume 3.0 cc; no sperm seen on routine analysis. Centrifuged pellet reveals no motile sperm.

The indications for obtaining a testis biopsy have expanded significantly over the last several years as a result of ICSI. Requiring only a single, viable spermatozoon per oocyte for microinjection, ICSI may be used with sperm derived from any source, including those found in testis biopsy specimens. As a result of these sperm harvesting indications, the testis biopsy now has new and important diagnostic and therapeutic implications for the infertile male (Table 1). For a description of surgical technique, the reader is referred to a current review by Coburn et al. in 1996. This purpose of this syllabus is to focus on advances and current concepts in testicular biopsy.

The urologist previously used the testis biopsy for diagnostic purposes only, i.e., to distinguish testicular failure, also known as non-obstructive azoospermia, from obstruction in the azoospermic patient. Now, sperm harvesting has become an important addition to the indications for this procedure. Still, the pivotal role of the biopsy in differentiating normal spermatogenesis from hypospermatogenesis, maturation arrests, and the Sertoli-cell-only states remains the most common and important indication for obtaining a testis biopsy. Much less commonly, testis biopsies may also be considered in the severely oligozoospermic (<1 million sperm/mL) male, if testicular size is relatively normal, to exclude a partial obstruction within the male reproductive tract, e.g., at the ejaculatory duct.

Prior to ICSI, the patient with testicular failure was told that adoption or the use of donor sperm was the only options available. Previous conventional teaching stated that azoospermic men with serum FSH concentrations more than 2–3 times normal had severe testicular failure not amenable to any conventional therapy. Therefore, a diagnostic testicular biopsy was considered unnecessary in these patients, especially if testicular atrophy was present. For these men, even if a single sperm could be found in 30% of their routinely processed hematoxylin and eosin stained sections, there was no chance that the gentleman could initiate his own biologic pregnancy with the then available methods of in vitro fertilization (IVF). However, these teachings were formulated prior to the introduction of ICSI. In contrast, the patient with an obstruction was offered a microsurgical reconstruction as the only possible method of initiating a pregnancy. With sperm harvesting for use with ICSI now being a reality, these men are all candidates for testis biopsy.

Generally, for the purposes of counseling the couple and to estimate the size and location of tissue needed for a sperm retrieval procedure, current practice is to perform a small diagnostic testis biopsy prior to proceeding with testicular sperm extraction (TESE), unless an obstruction is obviously present. If ICSI is an acceptable option to the couple, a testis biopsy may now be recommended to all azoospermic men with testicular failure. Using TESE, which is the isolation and extraction of sperm after a testis biopsy, Tournaye et al. recovered sperm in 81% of 54 TESE procedures. Specifically, a sperm recovery rate of 84% in patients with incomplete germ cell aplasia and maturation arrest, 76% with complete germ cell aplasia or maturation arrest, and 100% with hypospermatogenesis were reported. When these sperm were used for ICSI, a normal fertilization (2PN; 2 pronuclei) rate of 45.3% with an overall pregnancy rates of 33% per TESE procedure was observed. Although follow-up on the offspring is limited, no increased incidence of congenital abnormalities has been noted in these children. However, adequate counseling regarding the potential for genetic defects should be provided to the couple.

Similarly, using a single large testis biopsy of 450–500 mg of tissue, compared to the usual 50–100 mg for a routine diagnostic biopsy, Schlegel et al. were able to extract sperm in 10 of 16 (62%) TESE attempts, despite 30% of the cases having failed a previous TESE procedure. With a 2PN fertilization rate of 52%, biochemical pregnancies were achieved in 6/16 (38%) couples with a live delivery rate of 25%. In preparation for a possible TESE procedure, Turek et al. have described diagnostic testicular mapping of sperm using multiple percutaneous fine-needle aspirations (FNA). Using matched testicular fine-needle aspirates and open testicular biopsy specimens, they found that 4 of 12 men with nonobstructive azoospermia had localized "patches" of sperm detected in areas distant from sperm-negative biopsy sites. They concluded that for sperm detection, FNA can localize areas of sperm production within the testis and accurately guide sperm extraction procedures in men with nonobstructive azoospermia.

The ability to find sperm in the testis biopsies of men with testicular failure is clearly related to the heterogenous nature of spermatogenesis (Fig. 1) and sampling error inherent in the procurement of the small, standard biopsy specimen. Small, focal areas of spermatogenesis may be identified even in the presence of overwhelming testicular failure. Perhaps even more importantly, the ability to find sperm requires extreme patience, diligence, and tissue preparation expertise on the part of the person processing the tissue.

Even more successes are evident using sperm harvested from the testis biopsies of men with an obstruction and normal spermatogenesis. However, procuring sperm from the epididymis is advisable because of a higher yield of motile sperm, unless severe epididymal scarring is present. Tournaye et al. successfully recovered sperm in all 70 (100%) TESE procedures and had a normal fertilization and pregnancy rate of 62.5% and 43.5%, respectively. Instead of the standard open testis biopsy, percutaneous needle biopsy or percutaneous FNA may also be used with high sperm recovery rates. Because pregnancies have also been reported using frozen testicular sperm in men with obstructive azoospermia, an increasing practice is to cryopreserve a portion of the biopsy specimen for potential later use with ICSI. The long-term results of using sperm from frozen tissue aliquots remains to be reported.

Another proposed rationale for performing testicular biopsy in the oligospermic male relates to the issue of partial ductal obstruction. It has been suggested that this entity may result in oligozoospermia and that certain histological features may support this diagnosis. In 1981, Silber and Rodriguez-Rigau presented evidence that severe oligozoospermia can result from partial or subclinical epididymal obstruction. A relatively simple, quantitative analysis technique whereby the number of mature spermatids per seminiferous tubule is related to the sperm density in the ejaculate was suggested as a method of verifying partial ductal obstruction. In addition, in cases of persistent severe oligozoospermia after surgical vasectomy reversal, biopsy may also be appropriate to provide histological evidence for partial obstruction prior to performing secondary microsurgical reconstruction.

Figure 1. An algorithm for sperm procurement decision-making. In the non-obstructed azoospermic patient, testicular sperm extraction is the current procedure of choice, although experience with percutaneous methods is increasing. The author’s preference for most obstructed patients is to perform a microsurgical epididymal sperm aspiration, although a percutaneous sperm aspiration or testicular retrieval methods may be considered.

Several authors have observed an entity of partial ductal obstruction resulting from abnormalities of the ejaculatory duct, leading to severe oligozoospermia (usually accompanied by asthenospermia and decreased ejaculate volume), and remediable by transurethral resection of the ejaculatory ducts. Thus, it has often been our practice to perform testicular biopsy in severely oligospermic men with sperm densities of less than 1,000,000 sperm/mL when normal testicular size or mild atrophy is present. A normal testicular biopsy in such patients should prompt further diagnostic evaluation with a transrectal ultrasound to delineate any ejaculatory duct lesion.

As a result of these new indications for performing a testis biopsy, these practical questions arise:

(1) How many biopsies to obtain?

(2) How to determine when enough tissue has been obtained?

In the case of normal spermatogenesis, for sperm harvesting or diagnosis, only a single small biopsy from one testis is required. When testicular failure is present, however, a larger sampling of tissue is necessary. One approach for sperm harvesting is to take a single large biopsy. In contrast (for harvesting purposes), Tournaye obtained smaller individual samples from multiple sites, averaging 2.8± 2.5 biopsies (range 1–12) in Sertoli-cell-only, 4.2± 4.5 biopsies (range 1–20) in maturation arrests, and 1.5± 0.8 biopsies in hypospermatogenesis. The need for several testicular specimens, often from both testes, increases the likelihood of successful sampling due to intratesticular testicular histologic variability. The surgeon must realize and inform the patient that multiple biopsies or a single large biopsy may potentially, although rarely, damage the subtunical arterial supply to the testis, risking further testicular atrophy.

Proponents of multiple core biopsies of the testis, rather than a single superficial sampling, argue that their deeper sampling may have a better yield by providing a greater cross-sectional tissue core. These are most often performed in patients in whom asymmetrical lesions are suspected. Such suspicions may be aroused by disparity in testicular size, a unilaterally nonpalpable vas deferens, or by a history of prior scrotal, groin, or pelvic surgery. Bilateral biopsies could be useful in such cases to rule out obstruction of the excurrent ducts of one testicle; the sperm in the ejaculate might be arising from the contralateral, often smaller testicle with intrinsic dysfunction. Still, unilateral testicular biopsy provides adequate information in most clinical settings. A review of 192 infertile men showed that unilateral biopsy provided adequate assessment of the type and degree of testicular damage when a bilaterally symmetrical process was anticipated. When unilateral biopsy is to be performed, we generally choose the testicle that is grossly more normal in size, consistency, and shape.

When multiple biopsies are obtained for nonobstructive azoospermia, performance of a wet preparation or testicular touch preparation cytology intraoperatively are necessary to judge whether sperm are present. Once sperm are identified, the procedure may be terminated with a reasonable degree of certainty that more sperm will be found with further tissue extraction. Because these analyses take time, especially if no sperm are initially found, consideration should be given towards providing adequate sedation or general anesthesia for patient comfort.

Although not a new technique, the "touch imprint" (also known as testicular touch preparation cytology of a testis biopsy specimen) is an underutilized procedure. While routine testis biopsy with hematoxylin and eosin (H&E) staining is the generally accepted procedure for the evaluation of spermatogenesis, a limitation is its inability to consistently differentiate between a late maturation arrest (mature sperm not formed, but immediate precursors are present) and normal spermatogenesis. The ability to differentiate quickly and reliably between these two conditions in the azoospermic male at the time of potential microsurgical correction of ductal obstruction is another indication for performance of touch imprint cytology and should be mandatory.

While hematoxylin-eosin or Papanicolaou staining typically have been used after sending the cytofixed specimen to the frozen section pathology laboratory, a Diff-Quik (Baxter Healthcare Corporation, Miami, FL) staining method has recently been described. The advantages of this stain over H&E and Papanicolaou stains are: (1) that slides are allowed to air-dry first without a need for fixative, and (2) that skills of histologic technicians are not required, as this method can be performed in the operating room by circulating staff. Similar to the other staining methods, simple and rapid identification of spermatozoa and spermatids is possible.

In performing a touch preparation cytology, an open biopsy specimen is touched to or gently moved across a microscopic slide with fine tissue forceps (Fig. 2). Alternatively, the slide may be touched to the cut surface of the testicular parenchyma after excision of the biopsy specimen. The slide is immediately sprayed with cytofixative or immersed in 95% ethanol before air-drying can occur and stained with the Papanicolaou or hematoxylin and eosin techniques (Fig. 3). Cellular architecture is distorted if the slide dries before cytofixative is applied. The use of "touch preparation" cytology with a percutaneous ASAP core biopsy system (Travenol Laboratories, Deerfield, IL) in comparison with open biopsy diagnosis and open biopsy "touch preparation" cytology has also been described and demonstrated an excellent correlation.

These cytological methods provide a rapid means of examining the cellular contents of the seminiferous tubules. This quick diagnosis is especially important if microsurgical repair of an obstruction is to be performed concurrently. Because mature, intact spermatozoa are clearly identified in this manner, we have found such cytological preparations useful in distinguishing examples of late maturation arrest from normal biopsy specimens, a distinction that sometimes may be difficult on frozen or paraffin sections in which mature sperm are difficult to identify conclusively. In the azoospermic patient, we also have found touch imprint cytology to be more rapid and informative than the frozen section technique in confirming intraoperatively that mature sperm are present in the biopsy. The frozen-section technique has the added disadvantage of introducing distortion in the paraffin sections subsequently prepared from the frozen material.

Testicular Needle Biopsy. There has been increasing interest in recent years in the relative merits of percutaneous needle biopsy compared to open surgical testicular biopsy, and many reports on this topic have appeared. The proponents of needle biopsy suggest that this technique can be performed as an office procedure with little pain and low morbidity and yield adequate information in selected patients. Techniques described have included the use of the Vim-Silverman or TruCut® large needles to obtain a core of tissue for histological sections. Recently, the use of the Biopty gun® has been described as a simple means of obtaining a fine-core biopsy under local anesthesia.

All of these needle biopsy techniques involve the limitation of providing relatively few tubular cross-sections for examination, with the resultant loss of some histological information as compared to a standard open surgical biopsy specimen. These needle biopsy techniques have been described as being best suited for the azoospermic patient as a simple means of documenting spermatogenesis in the office setting, so that subsequent operative exploration with microsurgical capability can be planned. While further studies comparing needle biopsies with concurrent standard surgical biopsies are needed to determine the accuracy of needle biopsy techniques in quantifying spermatogenesis, a recent report has described a good correlation between percutaneous and open surgical biopsy specimen analysis. Kessaris and co-workers described a 95% correlation between percutaneous needle and open biopsy techniques in 24 testes (19 patients) in whom both techniques were applied, both for histologic assessment and touch imprint interpretation.

FNA cytology of the testis has also been described extensively and has been proposed to be a minimally traumatic procedure with good correlation with histologic studies. Performed with either a general anesthesia or a local anesthetic, a 21–23 gauge needle may be used with a 10–20 mL syringe. Gottschalk-Sabag has noted that a 23 gauge needle may not always provide sufficient material for cytologic analysis.

Through a single puncture site most commonly on the mid-testis and away from the epididymis, several aspirating passes are made. The anterior-lateral surface is preferable in order to avoid the testicular arterial supply. The aspirate is then sprayed onto slides, which are processed according to need. Air-drying is used for image analysis and prior to May-Grunwald-Giemsa staining, while immediate 95% EtOH fixation is used for the Papanicolaou stain. A propidium iodide rinse may be used for flow cytometry. Direct finger compression provides hemostasis.

Because of the "heterogenous picture" of the spermatogenic process within the testis, a single aspirate or biopsy may not be truly reflective of the entire testis. Given the small amount of material obtained, especially from aspirates, as least two separate sites have been recommended as necessary for FNAs. In one study, FNAs were obtained from three separate sites in 13 patients, resulting in a wide deviation in cytologic assessment from the various sites.

Testicular FNA has not gained widespread acceptance in the evaluation of the infertile male for numerous reasons. Although cellular detail is excellent, information regarding peritubular fibrosis, the interstitial tissue, and cellular arrangement is lacking. Distinguishing between hypospermatogenesis and obstruction may be difficult, if not impossible, with aspiration cytology alone, because of quantification issues. Concerns about severe testicular trauma and hematoma formation, although quite low in incidence, are also present. Finally, a lack of experienced testicular cytopathologists may also be contributory.

The evaluation of the testis biopsy specimen in male infertility has been hampered by the qualitative rather than quantitative nature of routine histologic interpretation. Manual quantitative methods were initially investigated but were extremely labor-intensive and never gained popularity.

More recently, DNA flow cytometry has been used in an attempt to quantify cell types, but has the significant disadvantage of not being able to distinguish between specific types of 1N cells, the spermatozoa and spermatids. Flow cytometry can demonstrate the typical DNA histogram of adult testicular tissue consisting of three major peaks: haploid (1N); diploid (2N) Sertoli and Leydig cells, secondary spermatocytes, and spermatogonia; and tetraploid (4N) primary spermatocytes during the meiotic process (Fig. 3). DNA image analysis can provide objective quantification of cell types from testicular aspirates or from paraffin-embedded, Feulgen-stained sections and is frequently used for determining the DNA content and cell cycle analysis in malignancies (Fig. 4).

By digitalizing microscopic images of a cell and computer analyzing their DNA content (picograms of DNA) and morphologic features (area, shape, size), image analysis is able to differentiate between spermatozoa and spermatids, as well as identify 2N and 4N cells. In this technique, Feulgen staining (DNA Staining Kit; Cell Analysis Systems, Inc., Elmhurst, IL) is performed on 5 µm recut sections of Bouin’s fixed, paraffin-embedded tissue. The Feulgen stain specifically and stoichiometrically binds to nuclear DNA, which is spectrophotometrically analyzed and quantitated using light microscopy at 400X and the CAS 200 Image Analysis System (Cell Analysis Systems, Inc, Elmhurst, IL). A specially designed testis biopsy filter based on cellular DNA content (picograms), area (m m²), shape (no units), and density (no units) has been developed to allow for differentiation of spermatozoa and spermatids. Those nuclei corresponding to settings in the testis biopsy filter are observed in situ on a video screen. Results are recorded as percentages of spermatozoa, spermatids, 2N, and 4N cells (Tables 2&3).

This system has important implications in redefining our current purely qualitative testis biopsy interpretation, and could replace or add an additional classification to this present system. Previous quantitative techniques never gained popularity because of their extremely labor-intensive nature. By providing a percentage of cell types present in a specimen, often misinterpreted terminology such as "partial," "incomplete," and "early" arrest becomes obsolete. The research applications are also significant, providing a quantifiable way of measuring results of a given future intervention, such as gene therapy, in azoospermic men.

A	B
Figure 4. A, Feulgen-stained testis biopsy specimen of normal spermatogenesis. B, After image analysis. The various colors represent different cell types. (From Kim E.D, et al: J Urol 157:147–150, 1997.)

In summary, as a result of ICSI, the indications, specific information required from a testis biopsy, and performance of the biopsy technique have changed significantly. Testis biopsies are now commonly being performed for therapeutic purposes, and finding even a rare mature spermatozoon has new importance. Because the majority of patients with clinically apparent testicular failure may harbor sperm within the testis, a testis biopsy should be offered to these men if ICSI is an acceptable alternative for the couple.