Journal of Andrology
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Journal of Andrology, Vol. 26, No. 2, March/April 2005
Copyright © American Society of Andrology

The Utility of Optical Loupe Magnification for Testis Sperm Extraction in Men With Nonobstructive Azoospermia

JOHN P. MULHALL, SAMEH W. GHALY, NADID AVIV AND ABSAAR AHMED

From the Departments of Urology, Loyola University Medical Center and Stritch School of Medicine, Maywood, Illinois.

Correspondence to: Dr John P. Mulhall, Department of Urology, Weill Medical College of Cornell University, New York Presbyterian Hospital, 525 E 68th St, Starr 900, New York, NY 10021.
Received for publication July 14, 2004; accepted for publication September 20, 2004.

   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
The testis of patients with nonobstructive azoospermia (NOA) harbors sperm in approximately 30% to 60% of cases. Use of an operating microscope has been shown to result in better sperm retrieval rates. This investigation was undertaken to evaluate the ability of a modified microsurgical approach using magnifying loupes (3.5x) to improve the rates of sperm retrieval during testis sperm extraction (TESE). The study group consisted of patients with NOA who underwent TESE. Before December 1998, TESE was conducted in a standard fashion, and from 1999 on, loupe magnification was used. Comparison was made between the 2 groups with regard to sperm retrieval rates, need for bilateral TESE, and number of tunical incisions. Overall sperm retrieval rates did not differ between the 2 groups (45% vs 50%). However, in patients with testicular volumes of 10 mL or less, patients who underwent standard TESE had a retrieval rate of 27% compared with 42% when using the optical loupe magnification (P = .025). The use of loupe magnification may permit surgeons without access to or experience using an operating microscope to obtain better rates of sperm retrieval in men with NOA who have testicular volumes of 10 mL or less.

     Key words: Male infertility, intracytoplasmic sperm injection


Azoospermia, which is the absence of spermatozoa from a centrifuged semen specimen, accounts for approximately 10% of all male infertility (Jarow et al, 2002). Azoospermia is classified as either obstructive or nonobstructive, the former resulting from an obstructing lesion in the extratesticular ductal system and the latter due to a failure of spermatogenesis. Nonobstructive azoospermia (NOA) accounts for approximately three quarters of all men who present with azoospermia (Jarow et al, 2002). The diagnosis of NOA is based on patient history, physical examination, hormone parameters, non-sperm semen parameters, and testicular histological features (Pierpaoli and Mulhall, 1999). The introduction of intracytoplasmic sperm injection (ICSI) has revolutionized the treatment of men with NOA (Palermo et al, 1992; Schlegel et al, 1997; Hourvitz et al, 1998; Pierpaoli and Mulhall, 1999). It is well recognized that the testis of patients with NOA is a reservoir for spermatozoa in approximately 30% to 60% of cases using testis sperm extraction (TESE) (Mulhall et al, 1997; Ben-Yosef et al, 1999; Chan and Schlegel, 2000a, 2000b).

It is also appreciated that spermatogenesis within a testis is a patchy phenomenon, with some foci of seminiferous tissue yielding spermatozoa, whereas others remain barren. Historically, a threshold volume of seminiferous tissue needed to be extracted to yield enough spermatozoa for multiple cycles of ICSI. Recent work has suggested that using an operating microscope may allow harvesting of a smaller quantity yet better quality tissue, resulting in higher sperm retrieval rates (Schlegel, 1999). Lack of familiarity with and problems with access to the use of an operating microscope result in some urologists who perform TESE continuing to perform this procedure in a macroscopic fashion. This investigation was undertaken to evaluate the ability of a modified microsurgical approach to improve the rates of sperm retrieval during TESE in patients with NOA.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 Conclusion
 References
 
Study Population

Patients with NOA, whose partners were due to undergo ICSI, underwent TESE. Patients were evaluated by comprehensive history and physical examination of at least 2 semen analyses (separated by at least 2 weeks) and hormone parameters, including serum total testosterone and follicle-stimulating hormone levels. Of importance, the assessment of testicular volume was conducted by a single clinician (J.P.M.) using a calipers combined with a Prader orchidometer. Testicular volumes for both testes were averaged and are presented as the mean volume. Before surgical sperm extraction, all patients underwent karyotype analysis and an evaluation for Y chromosome gene microdeletions.

Standard TESE

Before December 1998, all TESE procedures were conducted in a standard fashion (group 1) as follows: a 1-cm transverse skin incision was made over the testis and the tunica vaginalis was opened without delivering the testis. The tunica albuginea of the testis was incised with an eyelid retractor and seminiferous tissue was extruded. An intraoperative wet preparation was conducted, and if sperm were present within the first specimen, the procedure was terminated. In cases where no sperm were seen, the testis was rotated without delivery and a second tunical incision and tissue extraction were performed on the same side. Where this was not possible or when the second biopsy result was also negative for sperm on wet preparation analysis, an identical procedure was conducted on the opposite testis. Thus, patients underwent the extraction of at least 1 piece of seminiferous tissue and at maximum had the extraction of 4 separate pieces of seminiferous tissue. A portion of the extracted testis tissue was sent for formal histopathological analysis to determine the spermatogenic pattern and to assess the presence of intratubular germ cell neoplasia.

Optical Loupe Magnification Technique

From 1999 on, the standard technique was modified to incorporate loupe magnification (3.5x). Other than the use of magnification, an identical protocol was used. Magnification was used in an effort to permit identification of the healthiest-looking seminiferous tissue in an effort to reduce the number of harvest sites, reduce the need for bilateral TESE, and increase the overall sperm retrieval rates. The testis access and approach were otherwise identical; again no testis delivery was performed. Following incision of the tunica albuginea of the testis, the fullest seminiferous tubules were identified and harvested for sperm extraction.

Statistical Analysis

For comparison between the 2 groups as a whole, Fisher's exact test was used for comparison of continuous variables, and the {chi}2 test was used for categorical variables. Patients were subdivided into groups based on testicular volumes, and the Mann-Whitney U test was used for comparison of the subgroups.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 Conclusion
 References
 
Ninety-two consecutive patients with NOA were analyzed, including the last 44 patients who underwent standard TESE and the first 48 patients who underwent microdissection TESE at this institution. Patient demographic data and sperm retrieval rates are presented in Tables 1 and 2. No statistically significant difference existed between the 2 groups (standard TESE vs microdissection TESE) in patient age, histological pattern, and presence of AZFc gene deletion or chromosomal abnormalities. Of note, at the time of this analysis, AZFa and AZFb deletions were not probed for, and therefore no assessment of these deletions or combined deletions were made. No patient on histopathological analysis of the testis tissue had normal spermatogenesis and none had any intratubular germ cell neoplasia diagnosed. Of note, histological pattern was not a predictor of sperm retrieval rates within either group.


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  		Table 1. Patient demographics*
 

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  		Table 2. Sperm retrieval results*
 

Overall sperm retrieval rates did not differ statistically between the 2 groups (45% vs 50%). However, in patients with testicular volumes of 10 mL or less, patients who underwent standard TESE had a retrieval rate of 27% compared with 42% of those who underwent TESE by the microsurgical approach (P = .025). In the microdissection TESE group, there was also a decreased need for bilateral sperm extraction (42% vs 82%) and also a smaller number of tunical incisions or biopsies were needed (1.4 ± 0.4 vs 3.2 ± 1.2), both of which were statistically significant.


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
Conclusion
 References
 
The advent of ICSI has allowed the application of assisted reproductive technologies to couples whose male partner can produce only tiny numbers of sperm (Palermo et al, 1992; Van Steirteghem et al, 1993, 1994, 2002; Palermo et al, 1998; Bonduelle et al, 1999). This has particular relevance to patients with NOA (Mulhall et al, 1997; Oates et al, 1997; Schlegel et al, 1997; Schulze et al, 1997; Pierpaoli and Mulhall, 1999). Numerous retrieval techniques have been reported, with the general consensus being that open techniques have a higher likelihood of finding spermatozoa and in greater numbers than percutaneous approaches (Friedler et al, 1997; Mercan et al, 2000). Friedler et al (1997) analyzed the results of TESE in 37 men with rigorously diagnosed NOA. In this study, each patient underwent a percutaneous retrieval before an open sperm extraction at the same sitting. The retrieval rates were 11% for the percutaneous and 43% for the open approach.

In the standard TESE technique, the procedure is performed in a blind fashion that does not identify the focal sperm-producing areas of the testis until tissue has been excised from the patient. Microdissection of testicular tubules identifies sperm-containing regions before their removal. Identification of spermatogenically active regions of the testicle is possible by direct examination of the individual seminiferous tubules. The underlying concept for this technique is simple: seminiferous tubules that contain many developing germ cells, rather than Sertoli cells alone, are likely to be larger and more opaque than tubules without sperm production. In a sequential series of TESE cases for men with NOA, Schlegel (1999) demonstrated that the ability to find spermatozoa increased from 10 (45%) of 22 men to 17 (63%) of 27 men after introduction of the microdissection technique. Microdissected samples yielded an average of 160 000 spermatozoa per sample in only 9.4 mg of tissue, whereas only 64 000 spermatozoa were found in standard biopsy samples that averaged 720 mg in weight. For men in whom microdissection was attempted, successful identification of enlarged tubules was possible in 15 (56%) of 27 cases. Although the number of patients in this study was small, these findings suggested that microdissection TESE using optical loupe magnification is associated with improved sperm retrieval for men with NOA over that achieved with previously described biopsy techniques.

Amer et al (2000) evaluated the value of TESE by microdissection compared with open, classic surgical biopsy in the same patients. A total of 100 patients with NOA and bilateral identical testicular histological findings underwent bilateral diagnostic TESE via the conventional method on one side and the microsurgical method on the other side. The spermatozoa recovery rate by microdissection TESE was significantly higher than by conventional TESE (47% and 30%, respectively; P < .05). In another study, Schlegel and Su (1997) evaluated the effects of a standard TESE technique on the testis. Sixty-four patients were evaluated after TESE for NOA with physical examinations, serial scrotal sonography, histological analyses, and evaluation of the success of additional sperm retrieval attempts. Three months after TESE, 82% of patients had ultrasound-documented abnormalities in the testis, suggesting resolving inflammation or hematoma; however, by 6 months, these acute inflammatory changes typically resolved, leaving only linear scars or calcifications.

The standard protocol has been to retrieve a fresh sample of testis tissue on the day of oocyte recovery. Unfortunately, approximately 30% of men will possess no spermatozoa in their tissue, making it impossible to conduct ICSI with the male partner's sperm. Amer et al (1999) conducted a retrospective study in an effort to identify predictors of successful recovery of testicular sperm from the patients with NOA and ICSI outcomes in such couples. There was no parameter that absolutely predicted the recovery of testicular sperm from patients with NOA.

In our study, overall sperm retrieval rates did not differ between the 2 groups (45% vs 50%). However, in patients with mean testicular volumes of 10 mL or less, patients who underwent standard TESE had a retrieval rate of 27% compared with 42% for those who underwent TESE with loupe magnification (P = .025). Furthermore, the rate of bilateral TESE rates was lower in the microdissection TESE group compared with the standard TESE group, indicating the increased likelihood of retrieving sperm from the first testis dissected with the modified microsurgical approach. The concept of patchy spermatogenesis is well accepted; that is, in men with NOA, although there are areas that are barren of spermatozoa, other areas harbor tiny numbers of fully formed spermatozoa that can be harvested and used for ICSI.

The crux of the TESE technique is the identification of the area or areas within the seminiferous tissue that have spermatozoa present. The use of magnification, especially an operating microscope, has been well documented to permit visualization of seminiferous tubules that are fuller and more likely to harbor spermatozoa (Schlegel, 1999; Chan and Schlegel, 2000; Chan et al, 2001). Using a less powerful form of magnification, we have shown that the use of magnification loupes was capable of improving sperm retrieval rates in men with small testes. The primary limitation of this study is the fact that this is a nonrandomized analysis and the standard and loupe magnification approaches were performed at different points. However, a single surgeon already experienced in TESE before the commencement of the study (J.P.M.) performed all procedures, offsetting some of this concern. The size of the populations studied is small, and this may under-mine the statistical analysis. However, based on the analyses conducted, there appears to be a statistical significance using loupe magnification, and at the very least, this should urge other investigators to explore a randomized controlled trial comparing loupe magnification and operating microscope-assisted TESE.

The data obtained from this study suggest that in men with marked diminishment in testicular volume, specifically those with mean testicular volumes of 10 mL or less, the use of another less powerful modality of magnification, that is, magnifying loupes, can increase sperm yield. This approach may permit urologists who are not familiar with the use of an operating microscope or those without access to an operating microscope to increase sperm harvesting success.


   Conclusion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 Conclusion
References
 
These data suggest that the use of microsurgical techniques may permit better rates of sperm retrieval in men with NOA or profound oligospermia who have mean testicular volumes of 10 mL or less. No significant difference in overall sperm acquisition rates was seen between standard TESE and microdissection TESE. There was also a decrease in the need for bilateral TESE in men who underwent microdissection TESE. This information may be useful to the clinician in planning the surgical approach to patients with NOA.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
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Bonduelle M, Camus M, De Vos A, et al. Seven years of intracytoplasmic sperm injection and follow-up of 1987 subsequent children. Hum Reprod. 1999; 14(suppl 1): 243 -264.

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