Relationship between classic histological pattern and sperm findings on fine needle aspiration map in infertile men

Maxwell V. Meng1, Imok Cha2, Britt-Marie Ljung2 and Paul J. Turek1,3

1 Departments of Urology and 2 Pathology, University of California San Francisco School of Medicine, San Francisco, California, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Systematic testis fine needle aspiration (FNA) mapping has been proposed as an adjunctive or alternative diagnostic procedure to biopsy to determine the presence of spermatozoa within infertile testes. This study related testis histology to the global presence or absence of spermatozoa in the same testes determined by FNA cytology. Testis biopsies and FNA mapping were performed in 87 infertile, azoospermic men. A mean of 1.3 biopsies and 14 FNA sites were taken per patient. Biopsies were assessed by recognized histological patterns of normal, Sertoli cell-only, hypospermatogenesis, early and late maturation arrest, sclerosis as well as mixed patterns that included at least two of these histologies. FNA cytological specimens were assessed for sperm presence by an experienced cytologist. Overall, spermatozoa were found by FNA mapping in 52% of patients. A comparison of histology and FNA findings revealed that pure patterns of Sertoli cell-only and early maturation were associated with a very poor likelihood of sperm detection (4–8%). In contrast, patients with other pure pattern histologies or mixed patterns had high rates of FNA sperm detection (77–100%). Similar to reported testicular sperm extraction (TESE) findings, sperm detection with FNA shows wide variation depending on testis histology. Unlike most TESE reports, however, some histological patterns generally reflect a more global testicular dysfunction and poorer likelihood of sperm identification, suggesting the possibility that these phenotypes have a genetic origin. Systematic testis sampling with FNA offers additional geographical information about spermatogenesis that routine biopsies lack and can further guide couple decision-making in severe male factor infertility.

Key words: azoospermia/histology/infertility/needle aspiration/testis


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
One of the most exciting therapeutic offshoots of intracytoplasmic sperm injection (ICSI) is the use of testicular spermatozoa to achieve pregnancies (Schoysman et al., 1993Go; Silber et al., 1995Go). Although testis sperm retrieval in cases of obstruction is not difficult, there is failure to obtain spermatozoa for ICSI in 25–50% of men with severe testis failure, termed non-obstructive azoospermia (NOA) (Kahraman et al., 1996Go; Tournaye et al., 1996Go; Mulhall et al., 1997Go; Schlegel et al., 1997Go; Tournaye et al., 1997Go). Several strategies are currently used to determine more accurately which men with NOA are candidates for ICSI, in an attempt to minimize emotional and financial costs associated with cancelled in-vitro fertilization (IVF) cycles. These include simultaneous diagnostic and therapeutic testis sperm extraction (TESE) with cryopreservation (Mulhall et al., 1997Go) and real-time, multi-biopsy approaches performed with either optical magnification (Schlegel et al., 1997Go) or classic testis biopsy (Tournaye et al., 1996Go). Our strategy has been to perform a diagnostic testis fine needle aspiration (FNA) map in the office prior to the IVF cycle to identify in which NOA patients, and exactly where, spermatozoa are to be found (Turek et al., 1997Go). With this approach, we prospectively identified candidates with spermatozoa for ICSI to reduce IVF cycle cancellations and to maximize testis parenchymal conservation in NOA men with atrophic testes (Turek et al., 1999Go). Prior studies have used testicular FNA primarily as a therapeutic tool in the retrieval of spermatozoa for ICSI (Reubinoff et al., 1998Go; Lewin et al., 1999Go).

It is clear from increasing experience with TESE procedures in NOA patients that recognized clinical parameters, including testicular size, prior history of ejaculated spermatozoa, serum follicle-stimulating hormone (FSH) concentrations, or testis biopsy histology, do not accurately predict whether or not spermatozoa will be recovered during testicular exploration (Kahraman et al., 1996Go; Mulhall et al., 1997Go; Schlegel et al., 1997Go; Tournaye et al., 1997Go). Among these clinical features, however, testis biopsy histology is the best predictor of sperm presence at TESE (Tournaye et al., 1997Go; Su et al., 1999Go). Thus, we compared our sperm findings from diagnostic testis FNA maps to those from testis histology to assess whether a relationship similar to that between histology and TESE findings could be identified with histology and FNA findings.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patient selection
FNA and testis biopsy procedures were performed in 87 consecutive infertile, azoospermic patients. A detailed history and physical examination was performed on all patients. Testis volume was estimated with a Prader orchiometer (ASSI, Westbury, NY, USA). In addition, a semen analysis, centrifuged pellet of ejaculate, and post-ejaculate urine were examined to confirm true azoospermia. Hormone evaluations including testosterone and FSH were obtained in all patients. This study was conducted with prior approval from the university institutional review board.

FNA technique
The FNA procedure was performed as previously described (Turek et al., 1997Go). Briefly, the scrotal skin was cleaned and the spermatic cord infiltrated with local anaesthesia. The testis was positioned with the epididymis directed posteriorly. The scrotal skin was stretched taut over the testis by wrapping the scrotal skin behind the testis with a sponge. The planned aspiration sites were marked on the scrotal skin overlying the testis. FNA was performed with a sharp-bevelled, 1 inch, 23 gauge fine needle (Becton-Dickinson Co., Franklin Lakes, NJ, USA) using the established suction-cutting technique (Ljung, 1992Go). A 10 ml syringe (Becton-Dickinson Co.) was placed on suction in a Cameco syringe holder (Precision Dynamics Corp., San Fernando, CA, USA). Precise, gentle, in-and-out movements, varying from 5–8 mm, were used to aspirate tissue. Ten to 30 needle excursions were made at each site. Suction was released, and the tissue was expelled onto a slide, gently smeared, and fixed immediately in 95% ethyl alcohol. Pressure was applied to each site after aspiration for haemostasis. A routine Papanicolaou stain was performed on the smear.

FNA interpretation
Each stained FNA cytological smear was interpreted by a single experienced cytopathologist for (i) the presence or absence of mature spermatozoa with tails, and (ii) specimen adequacy, as previously reported (Turek et al., 1997Go). An adequate, and therefore informative, FNA specimen was defined as that which contained at least 100 clusters of 20 or more cells or at least 2000 well-dispersed testis cells. FNA adequacy rates were closely monitored and are reported here.

Testis biopsy technique and interpretation
Among the 87 patients, 29 men had testicular biopsies performed prior to referral for FNA mapping and 58 men underwent concurrent biopsy and FNA. Formal histological review of all prior biopsies was undertaken by a single pathologist (I.C.). When performed concurrently with FNA, testis biopsies were obtained by the `window' technique (Coburn and Wheeler, 1991Go) under local anaesthesia after FNA sampling. A touch imprint of the biopsy tissue was made and the tissue then placed in Bouin's solution and processed for routine histology. Touch imprint data are not presented here.

Testis biopsies were stained with haematoxylin and eosin to visualize histological details and classified according to Levin (1979): normal spermatogenesis, germ-cell hypoplasia or hypospermatogenesis, complete (early) or incomplete (late) maturation arrest, germ-cell aplasia (i.e. Sertoli cell-only) and tubular sclerosis. If a biopsy contained a single histological pattern throughout the sample, it was considered a pure pattern. If biopsies with early maturation arrest, germ cell aplasia, or sclerosis showed complete spermatogenesis in any area of the specimen, then a mixed pattern was assigned. Since mature spermatozoa with tails were difficult to see on biopsy, the finding of late spermatids was interpreted as presence of spermatozoa. All testis biopsies were interpreted by a single pathologist (I.C.).

The histological difference between late maturation arrest and normal spermatogenesis can be difficult to discern. We used other clinical features, including physical examination, semen analysis, and hormone evaluation, to differentiate between the two. Therefore, patients with numerous spermatids on histology but without clinical evidence of ejaculated spermatozoa or confirmed obstruction were deemed to have late maturation arrest.

Relationship of FNA to biopsy findings
For each patient, the sperm findings from the FNA map and the testis biopsy were examined to determine relationships between histological diagnoses and the presence of spermatozoa by FNA. This relationship is similar to that used earlier (Tournaye et al., 1997Go) in which histological diagnosis was related to sperm detection by TESE. To account for biases due to biopsy and FNA sampling intensity, we calculated the mean number of biopsy or FNA samples taken from each patient. Simple descriptive statistics were applied when appropriate.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patient characteristics
Eighty-seven men underwent evaluation with testis biopsy and FNA mapping. The mean age was 36.9 years (SD ± 7, range 24.7–53.1) with a mean right testicular volume of 14.5 ml (SD ± 4.7) and a mean left testis volume of 14.4 ml (SD ± 4.2). The diagnoses associated with azoospermia in these patients are listed in Table IGo. Approximately 90% of patients were classified as NOA based on clinical, laboratory and histological information. Among the 7% of men with obstructive azoospermia, half had congenital bilateral absence of the vas deferens (CBAVD) and the other half had idiopathic obstructive azoospermia. Patients with CBAVD were biopsied to confirm spermatogenesis prior to planned sperm aspiration procedures.


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Table I. Clinical diagnoses and sperm findings associated with azoospermia
 
Sperm findings by diagnosis
Overall, spermatozoa were found by FNA mapping in 52% of all patients. Among men with non-obstructive azoospermia (n = 81) 47% had spermatozoa detected by FNA mapping. Patients with idiopathic non-obstructive azoospermia had the worst prognosis for sperm detection, while all other groups, both obstructive and non-obstructive, had at least a 69% chance of harbouring spermatozoa by FNA (Table IGo).

FNA and biopsy results
A mean of 14 fine needle aspirations were performed per patient. The overall FNA specimen adequacy rate was 98.6%; there were no differences in FNA adequacy rates among patients with different histological diagnoses. In those patients in whom spermatozoa were detected by FNA, a mean of 70% of different sites demonstrated spermatozoa. A mean of 1.3 testicular biopsies were performed/patient; there were no significant differences (by unpaired t-tests) in the number of biopsies taken among all histologies.

Pure histological patterns observed included normal spermatogenesis, hypospermatogenesis, germ cell aplasia (Sertoli cell-only), early maturation arrest, and late maturation arrest. Sclerosis was always found in conjunction with another pattern. Among the 29 men referred for FNA with a prior testis biopsy, none was normal: 19 men had pure patterns of either germ cell aplasia or early maturation arrest, and hypospermatogenesis and late maturation arrest were found in eight and two men respectively. Although normal spermatogenesis was confirmed in all six men with suspected obstruction, it was also observed in two men with idiopathic azoospermia, three with varicocele and one with cryptorchidism. The various histologies and the frequency with which they were detected are found in Table IIGo.


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Table II. Analysis of fine needle aspiration (FNA) map and biopsy procedures in azoospermic men
 
Relationship between histology and FNA findings
A close association existed between biopsy histology and sperm presence on FNA (Figure 1Go). Spermatozoa were detected in all patients with pure patterns of normal spermatogenesis, hypospermatogenesis, and late maturation arrest. In contrast, a single patient with early maturation arrest (1/13, 8%) and only one patient with a pure Sertoli cell-only pattern (1/28, 4%) had spermatozoa on FNA mapping. Interestingly, 10 of 13 men (75%) with mixed histological patterns demonstrated spermatozoa; of the patients without spermatozoa, one had a mixed pattern with Sertoli cell-only and tubular sclerosis and the other two had a mixed pattern of Sertoli cell-only and early maturation arrest. An an example, Figure 2Go illustrates the matched histological and FNA cytological findings on a patient with a mixed-pattern biopsy containing late maturation arrest and sclerosis. The association between biopsy and FNA findings was unrelated to the number of FNA sampling sites taken per patient.



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Figure 1. Association between biopsy histology and fine needle aspiration (FNA) mapping. Sperm presence was determined by FNA and histological patterns by open biopsy. Parentheses indicate the number of patients who exhibited the pattern. Hypo = hypospermatogenesis; SCO = Sertoli cell-only; EMA = early maturation arrest; LMA = late maturation arrest.

 



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Figure 2. (A) Testis biopsy histology showing mixed pattern consisting mainly of sclerosis and some tubules with late maturation arrest (upper left quadrant, arrows) (haematoxylin and eosin; scale bar = 200 µm). (B) FNA cytological smear from same patient. Note the presence of numerous small, darkly stained cells consistent with mature spermatozoa (arrow) (PAP stain; scale bar = 50 µm).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The development of ICSI is an exciting advance in the treatment of male infertility because it allows men with even the most severe forms of infertility the opportunity for paternity. It also presents clinicians with a new and difficult diagnostic challenge – to find spermatozoa within the failing, atrophic testes of candidate men with NOA. In an attempt to minimize the costs associated with cancelled IVF cycles, several strategies are currently used to determine more accurately in whom and where spermatozoa are to be found in NOA patients. Mulhall et al. have reported the use of simultaneous diagnostic and therapeutic TESE with cryopreservation (Mulhall et al., 1997Go) as a method to identify ICSI candidates. In men with NOA, this approach detected spermatozoa in 21 of 30 (70%) attempts. Tournaye et al. perform TESE at the time of egg retrieval with the multi-biopsy approach, and initially reported very high sperm recovery rates of 85% in 54 NOA patients (Tournaye et al., 1996Go); however, with more experience, the overall sperm-retrieval rate fell to 52.7% among 222 NOA patients (Tournaye et al., 1997Go). Schlegel and colleagues have proposed the use of the real-time, multi-biopsy approach performed with optical magnification to identify ICSI candidates (Schlegel et al., 1997Go). In a series of 16 men, spermatozoa were found in 10 (62%). In this study of diagnostic testis FNA maps, we located spermatozoa in 47% of men with NOA. Thus, by any of the various methods that have been described, many NOA patients can be observed to have retrievable, mature spermatozoa within their testes. At present, it is difficult to discern whether the observed differences in detection and retrieval rates among centres reflect true differences in technique or simply variations in patient numbers or clinical characteristics.

Close examination of the histological findings published in these TESE studies reveals a relationship not only between the classic histological pattern and sperm presence, but also between a pure or mixed histological pattern and sperm findings. A mixed histological pattern implies focality and heterogeneity to the testis parenchyma and may have stronger or different implications for sperm detection than globally uniform or pure patterns. In the series by Mulhall et al. (1997) the sperm retrieval rate in men with germ cell aplasia on histology was 50%; with maturation arrest or hypospermatogenesis (grouped), spermatozoa were found in 75% of patients. In addition, all men with late spermatids present on histology had spermatozoa recovered during TESE. An analysis of the biopsy findings from Schlegel et al. reveals that nine of 14 patients in the series had mixed histological pattern, and five had a single pattern on histology. Of the men with mixed histology, five of nine had spermatozoa retrieved; among the patients with pure histological findings, four of five had spermatozoa present, including one of two men with germ cell aplasia and both men with hypospermatogenesis (Schlegel et al., 1997Go). Early in their experience, Tournaye and colleagues found that sperm recovery was possible in 50% of men with pure germ cell aplasia and 82% of men with pure maturation arrest on histology. All men with a diagnosis of hypospermatogenesis had spermatozoa recovered at TESE (Tournaye et al., 1996Go). Further experience led to the observation that pure histological patterns on biopsy related to lower sperm retrieval success than mixed patterns: 19 versus 86% respectively, for germ cell aplasia and 48 versus 62% for maturation arrest (Tournaye et al., 1997Go). Thus, this large experience with TESE suggests that the fine details of testis histology may have important implications for sperm detection at TESE.

The sperm detection results from FNA mapping in this study suggest that even more striking relationships may exist between testis histology and sperm presence. The fact that only 4% of men with pure Sertoli cell-only histology and 8% of patients with early maturation arrest histology had spermatozoa on FNA map suggests that pure forms of these specific patterns may be more likely to reflect profound and global states of spermatogenic failure. Alternative explanations for the low FNA sperm detection rates with these biopsy patterns include the possibility that the FNA map technique is not as sensitive as other sperm detection methods or that there is significant variation in the attention given to the recognition of subtle biopsy patterns at different centres. Evidence against the lack of FNA sensitivity in sperm detection can be found on examination of the very high sperm detection rates (100%) associated with other pure histological patterns, including normal, hypospermatogenesis and late maturation arrest, rates similar to or exceeding those in the published TESE studies. The fact that biopsy interpretation varies by reader is certainly a well recognized and common phenomenon, although difficult to prove.

Biopsy patterns showing a mixed histology in which maturation arrest or Sertoli cell-only co-exists with a second pattern harbouring late spermatids uniformly showed spermatozoa on FNA. On the contrary, mixed pattern biopsies that harbour no spermatids represented testes without spermatozoa. Indeed, foci of spermatids within mixed pattern biopsies may be small (and therefore missed due to sampling error), but their significance is clear – if present in any portion of the biopsy, then spermatozoa will inevitably be found by FNA. This conclusion, also noted by Mulhall et al. in their TESE series (Mulhall et al., 1997Go), is corroborated by the spermatid findings from the pure pattern histologies as well.

The striking differences in FNA sperm detection rates from men with various global, pure-pattern biopsy histologies are far more pronounced than previously reported using TESE for sperm detection (Tournaye et al., 1996Go, 1997Go; Mulhall et al., 1997Go; Schlegel et al., 1997Go). If true, these differences may actually reflect the existence of real and profound physiological blocks to complete spermatogenesis. For instance, might a uniform, early maturation arrest pattern suggest an important meiotic block to the completion of spermatogenesis, as others have suggested (Vogt et al., 1992Go; Reijo et al., 1995Go)? Is it likely that a global pattern of Sertoli cell-only histology is more likely to reflect a disorder of genetic origin than a mixed histology biopsy partly harbouring this pattern? As experience accrues, it will be interesting to observe whether Y chromosome microdeletions and other, yet to be discovered, genetic links with male infertility associate strongly with certain pure pattern phenotypes of testis histology.


    Notes
 
3 To whom correspondence should be addressed at: Department of Urology, U-575, University of California San Francisco, San Francisco, CA 94143–0738, USA. E-mail: mrvas{at}itsa.ucsf.edu Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on December 24, 1999; accepted on June 6, 2000.