fna mapping fna mapping fna mapping is an application of fine-needle aspiration (fna) to the testis for the diagnosis of male infertility. fna cytology has been used to examine pathological human tissue from various organs for over 100 years. as an alternative to open testicular biopsy for the last 40 years, fna mapping has helped to characterize states of human male infertility due to defective spermatogenesis. although recognized as a reliable, and informative technique, testis fna has not been widely used in u.s. to evaluate male infertility. recently, however, testicular fna has gained popularity as both a diagnostic and therapeutic tool for the management of clinical male infertility for several reasons: the testis is an ideal organ for evaluation by fna because of its uniform cellularity and easy accessibility. the trend toward minimally invasive procedures and cost-containment views fna favorably compared to surgical testis biopsy. the realization that the specific histologic abnormality observed on testis biopsy has no definite correlation to either the etiology of infertility or to the ability to find sperm for assisted reproduction. assisted reproduction has undergone dramatic advances such that testis sperm are routinely used for biological pregnancies, thus fueling the development of novel fna techniques to both locate and procure sperm. for these reasons, there has been a resurgence of fna as an important, minimally invasive tool for the evaluation and management of male infertility. changes to infertility medicine advances in assisted reproductive technology (art) have revolutionized the ability to help men with even the severest forms of male infertility to become fathers. this field began in earnest in 1978 when the first successful in vitro fertilization (ivf) cycle was performed. this technique involves controlled ovarian stimulation followed by egg retrieval, in vitro fertilization, and embryo transfer to the uterus. in the united states, the number of babies born to infertile couples with ivf has risen logarithmically from 260 babies in 1985 to almost 50,000 in 2003. another significant advance in art was the development of intracytoplasmic sperm injection (icsi) in 1992. performed in conjunction with ivf, icsi involves the injection of a single, viable sperm directly into the egg cytoplasm in vitro to facilitate fertilization in cases of low sperm numbers. icsi has decreased the numerical sperm requirement for egg fertilization with ivf from hundreds of thousands of sperm for each egg to a single sperm. in addition, icsi allows sperm with limited intrinsic fertilizing capacity, including "immature" sperm obtained from the reproductive tract of men with no sperm in the ejaculate to reliably fertilize eggs. indeed, icsi has become so popular that u.s. clinics have routinely used it in more than 56% of their ivf cases. the success of icsi has also encouraged reproductive clinicians to look beyond the ejaculate and into the male reproductive tract to find sperm. currently, sources of sperm routinely used for icsi include sperm from the vas deferens, epididymis, and testicle. as art has evolved, so too have novel fna techniques to help diagnose and treat severe male infertility. an example of this is use of testicular fna "mapping" to systemically assess and localize sperm for art in men with azoospermia (no sperm count) and with testis failure characterized by "patchy" or "focal" spermatogenesis. indeed, this combination of techniques has allowed men with even the severest forms of infertility, including men who are azoospermic after chemotherapy for cancer, to become fathers. accuracy from a contemporary search of the english literature, it is apparent that the diagnostic testis biopsy has been used to study the pathological basis of male infertility for 60 years. the surgically obtained testis biopsy accurately describes testis architecture, is the best technique to detect in situ neoplasia or cancer, and allows for the overall assessment of the interstitium (leydig cell number and hypertrophy). levin described a qualitative method of assessing testis histologic patterns that is commonly used clinically to assess testis pathology in male infertility. recognized patterns include: normal spermatogenesis, hypoplasia or hypospermatogenesis, complete or early maturation arrest, sertoli cell-only or germ cell aplasia, incomplete or late maturation arrest, and sclerosis. johnsen proposed a more quantitative analysis of testis cellular architecture based on the concept that testis damage causes a successive disappearance of the most mature germ cell type. the johnsen scoring system involves a quantitative assessment of individual germ cell types that is very detailed and relatively laborious for routine clinical use. regardless of methodology, the analysis of testis biopsy histology lacks clinical value in cases of infertility because there is no clear correlation between histologic patterns or johnsen score and the underlying etiology of infertility. that is to say, the clinical utility of understanding the histology pattern is low, because biopsy patterns do not correlate well with specific and correctable diseases. in addition, the interobserver variability in testis biopsy readings for infertility is significant. this was aptly demonstrated in a study by cooperberg et al. in which the histology readings from two independent pathological reviews were prospectively compared with 113 testis biopsies undertaken for infertility. importantly, in 28% of cases preparation artifact or insufficient biopsy size rendered the sample suboptimal for interpretation. in addition, in 46% of cases, the two reviews disagreed, and this discordance resulted in significant changes in clinical care in 27% of cases. the most common error in pathological review was the under-appreciation of mixed histological patterns that are common and characteristic of infertile men with no sperm counts. thus, although commonly used, the classical testis biopsy has little or no correlation with specific diseases, is associated with significant variability in interpretation, and can miss mixed patterns of spermatogenesis that may qualify infertile men for assisted reproduction. fna cytology vs testis biopsy in many countries, testicular fna cytology is preferable to surgical testis biopsy for the evaluation of male infertility. in addition to accurately demonstrating the presence or absence of mature sperm with tails, fna provides tubular cells for cytologic analysis, also informative for the diagnosis of infertility. unlike testis biopsy histology, however, fna cytology has not been rigorously evaluated for the ability to distinguish in situ neoplasia and testis cancer. despite this caveat, the correlation of fna cytology to testis biopsy histology is very high (84–97%) in comparative studies comprising almost 400 patients (table 1). table 1. literature that correlates testicular fna cytology to biopsy histology study# patients % agreement persson, 19714286% gottschalk-sabag, 19934787% mallidis, 19944694% craft, 19971984% odabas, 19972490% mahajan, 19996097% rammou-kinia, 19993087% meng, 20008794% qublan, 20023496% aridogan, 20034090% mehrotra, 20075894% similar to the issue with biopsy histology, although several excellent descriptions of testis seminiferous epithelium cytology have been reported, no individual classification method has been uniformly adopted by cytologists as a standard approach. papic et al. quantified each cell type and calculated various cell indices and ratios and found that cytologic smears correlated well with histologic diagnoses. verma et al. also determined differential cell counts in patients with normal spermatogenesis; however, the reported ratios differed dramatically from those of papic. batra et al. have also published a cytologic schema in which the spermatic index, sertoli cell index, and sperm-sertoli cell indices are categorized into various histologic groups. using this system, they reported that differences in cell counts and indices could predict histologic categories. among the different classification systems for testis fna, those that attempt to correlate cytologic findings with biopsy histology hold the most promise to replace histology. with the goal of replacing the more invasive biopsy histology with fna cytology, a simple, working classification schema for testis fna cytology has been proposed that is based on pattern recognition. the identified patterns represent histological diagnoses but are based on relative numbers of three easily identified germ cell types on cytologic assessment: primary spermatocytes, spermatids, and spermatozoa. the various germ and sertoli cells are not precisely counted but are loosely quantified by assessing the relative number of cells present. in this way, a standard ratio of cell types for comparison to sertoli cell density is created. the fna patterns for hypospermatogenesis, sertoli cell-only, early maturation arrest, and late maturation arrest show different ratios of these three cell types relative to normal spermatogenesis and are relatively easy to categorize in this way. in a review of 87 patients with paired fna maps and biopsy, this cytologic classification was observed to be reproducible and accurate. histologic categories determined by fna patterns correlated well with open biopsy histology across all patterns in 94% of cases. fna cytology was also able to completely describe mixed histologies in 12 of 14 cases. thus, the determination of histology by fna cytology pattern is accurate and suggests that the more invasive testis biopsy is unnecessary to diagnose states of infertility. alternatively, it is possible that newer processing methods can be applied to testis fna cytology specimens to concentrate and prepare seminiferous tubules for sectioning similar to classic histologic preparations. either way, testis cytology is a viable alternative to histology in the evaluation of male infertility. newer concepts: fna "mapping" it has become clear to reproductive urologists who regularly perform testis sperm retrieval in azoospermic men for ivf-icsi that spermatogenesis varies geographically within the testis. this underscores the limitation and inadequacy of a single, localized testis biopsy or a single fna specimen to accurately reflect the biology of the entire organ. in fact, the current clinical challenges are: (1) to determine which infertile men with azoospermia harbor sperm for ivf-icsi and (2) to precisely locate the areas of sperm production within atrophic, nonobstructed testes. this clinical need led to the development of testis fna "mapping" in male infertility by a team of university of california san francisco physicians led by paul j. turek (figure 1). figure 1. extended fna mapping template for men with nonobstructive azoospermia used to define the presence or absence of testicular sperm. this example shows 12 individual fna sites per testis. in a pilot study of 16 men from 1997, testis fna was used to "map" the organ for mature sperm. the concept for mapping testis for sperm was inspired by the work of gottschalk-sabag and colleagues and modeled after the approach to prostate biology in which multiple prostate biopsies are used to detect foci of prostate cancer. similarly, fna was applied systematically to detect the presence or absence of sperm in varied geographical areas of the testis. in this study, azoospermic men underwent simultaneous testis biopsies and site-matched fna, and fna was found to be more sensitive in detecting sperm, as several men had sperm found by fna but not on biopsy. additionally, in one-third of patients, localized areas of sperm were detected by fna in areas distant from biopsy sites without sperm. these data confirmed intratesticular heterogeneity with respect to sperm distribution and suggested the potential of fna to localize patches of active spermatogenesis in failing testes. testicular fna mapping is performed using classic fna technique. under local anesthesia in the office, the testis and scrotal skin are fixed relative to each other with a gauze wrap. the "testicular wrap" is a convenient handle to manipulate the testis and also fixes the scrotal skin over the testis for the procedure. aspiration sites are marked on the scrotal skin, 5 mm apart according to a template. the number of aspiration sites varies with testis size and ranges from 4 (to confirm obstruction) to 15 per testis (for nonobstructive azoospermia). fna is performed with a sharp-beveled, 23-gauge, one-inch needle using the established suction cutting technique. precise, gentle, in-and-out movements, varying from 5 mm to 8 mm, are used to aspirate tissue fragments. ten to 30 needle excursions are made at each site. suction is released, and then the tissue fragments are expelled onto a slide, gently smeared, and immediately fixed in 95% ethyl alcohol. pressure is applied to each site for hemostasis. a routine papanicolaou stain is performed. smears are reviewed by experienced cytologists for (a) specimen adequacy (defined as at least 12 clusters of testis cells or at least 2000 well-dispersed testis cells) and (b) the presence or absence of mature sperm with tails. for immediate interpretation, fixed slides are stained with undiluted toluidine blue and read with brightfield microscopy after 15 seconds. patients take an average of two pain pills after the procedure. complications in over 800 patients have included one episode of hematospermia and, in another patient, post-operative pain for 7 days. in another study, systematic testis