Testicular sperm extraction in azoospermic men submitted to bilateral orchidopexy

L. Negri1,3, E. Albani1, M. DiRocco2, G. Morreale1, P. Novara1 and P.E. Levi-Setti1

Departments of 1 Reproductive Medicine and 2 Anatomic Pathology, Istituto Clinico Humanitas, Via Manzoni 56, I-20089 Rozzano, Milan, Italy

3 To whom correspondence should be addressed. e-mail luciano.negri{at}humanitas.it


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: This study was carried out to evaluate whether bilateral orchidopexy represents a poor or good prognostic factor in azoospermic men undergoing testicular sperm extraction (TESE). METHODS: One hundred and seven presumed non-obstructive azoospermia (NOA) patients, according to conventional clinical parameters (volume of testis, FSH, clinical history) were submitted to testicular biopsy with TESE. Thirty men (28%) had a history of bilateral orchidopexy for cryptorchidism. RESULTS: Normal spermatogenesis or mild hypospermatogenesis was diagnosed in 12/30 ex-cryptorchid patients and in 7/77 presumed NOA patients (P = 0.0004). Conversely, pure Sertoli cell-only syndrome or complete maturation arrest was found in 10/30 ex-cryptorchid patients and in 48/77 presumed NOA patients (P = 0.0094). In 53/107 patients (49.5%), TESE allowed a positive sperm retrieval. At least one spermatozoon was observed in 22/30 (~73%) ex-cryptorchid patients and in 31/77 (~40%) presumed NOA patients (P = 0.0026). A large number of spermatozoa (equivalent to an obstructive pathology) were retrieved in 13/30 ex-cryptorchid and in 10/77 presumed NOA patients (P = 0.001). A history of bilateral orchidopexy in presumed NOA patients correlates positively for the chance of retrieving testicular spermatozoa (odds ratio 3.8; 95% confidence interval 1.41–10.21; P = 0.008). CONCLUSIONS: Although bilateral cryptorchidism is usually considered a testicular secretive dysfunction, TESE permits retrieval of a large number of spermatozoa in almost 40% of cases. Our data suggest the existence of congenital or acquired obstructive anomalies of the seminal ducts in azoospermic orchidopexed men.

Key words: azoospermia/cryptorchidism/male infertility/seminal duct obstruction/sperm extraction/testicular


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The recent application of ICSI using testicular spermatozoa has renewed the interest in azoospermiae (Ezeh, 2000Go; Sharif, 2000Go; Ezeh and Moore, 2001Go). Azoospermia is usually classified as obstructive or non-obstructive (NOA). Bilateral cryptorchidism (maldescended testes) is usually associated with a secretory dysfunction (Ezeh et al., 1999Go; Sharif, 2000Go), with altered semen parameters and low paternity rate (Lee and Coughlin, 2001Go). Almost 10% of infertile patients seeking medical assistance have a history of cryptorchidism (Mieusset et al., 1995Go) and azoospermia is present in 20% of cases. Bilateral cryptorchidism represents the phenotypical expression of a severe anatomical and functional defect of the gonads (Hadziselimovic, 1996Go) and most of these patients have low orchidometry, abnormally soft testes and elevated serum FSH (Nistal and Paniagua, 1997Go). Testicular biopsy shows a significantly lower Johnsen score compared with patients who did not have maldescended testes, and the probability of extracting spermatozoa from testicular samples [testicular sperm extraction (TESE)] seems low (Glander et al., 2000Go).

The aim of this retrospective study was to analyse whether bilateral orchidopexy represents a poor prognostic factor in azoospermic men undergoing TESE. Clinical and histological data plus TESE-ICSI results of a population of azoospermic men who underwent bilateral orchidopexy have been matched with those of NOA patients without previous history of maldescended testes.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients
The population comprised 107 consecutive azoospermic patients [mean age (SD): 34.7 ± 4.4 years] who referred to our infertility unit over a 2-year period. No patient had a testicular biopsy available. The patients were presumed to be affected by NOA when presenting at least one of the following eligible criteria (Ezeh et al., 1999Go): (i) high FSH level (93 cases); (ii) low orchidometry (larger testis <12 ml) (73 cases); (iii) previous chemotherapy or radiotherapy for malignant disease (four cases); (iv) bilateral orchidopexy (30 cases); (v) bilateral mumps orchitis (four cases); or (vi) bilateral torsion of the testis (one case). Chronic renal insufficiency, previous heroin use plus hepatitis C virus infection, severe obesity and very large varicocele were observed in single cases. In 64 subjects (~60%) no aetiopathogenetic factor could be retrieved from clinical history (idiopathic NOA).

Thirty men had a previous history of bilateral cryptorchidism. The mean (SD) age of the patients was 32.9 (4.2) years. Age at bilateral orchidopexy ranged between 1 and 19 years [mean (± SD) 7.3 ± 4.6] and precise testicular location was available only in some patients, although the scars of bilateral inguinotomy were visible in all subjects. Six patients were monorchid because of monolateral orchiectomy or post-surgical testicular atrophy. In eight cases, hormonal therapy had been unsuccessfully administered before surgical treatment. In all subjects, infertility was primary, ranging from 1 to 11 years. All patients had already undergone semen analyses and six had had sporadic spermatozoa in the past.

Non-invasive diagnostics
Azoospermia was reconfirmed after two semen analyses with an interval of 1–2 months, according to the World Health Organization (1999)Go. Cryptozoospermia and retrograde ejaculation were excluded. Sperm culture was performed on all 107 patients. All patients were queried regarding general diseases, and specific questions were asked relating to associated obstructive or secretory dysfunction.

Serum FSH was rechecked using chemiluminescent microparticle immunoassay (Architect, Abbott, IL, USA) (normal range 1.3–9.0 mIU/ml). Scrotal duplex scanner for a precise testicular volume determination was performed. Attention was paid for subclinical testicular cancer, microlitiasis and ectasia of the rete testis. Transrectal ultrasonography was performed on all patients according to the methods described by Colpi et al. (1997)Go. Referring to genetic screening, all patients were evaluated with karyotype examination of peripheral leukocytes, cystic fibrosis gene mutation screening and Y chromosome microdeletions.

Scrotal exploration and TESE procedure
All patients gave written consent for these procedures. Open testicular biopsy (TB) was performed in day-surgery clinic under local anaesthesia. A prophylactic antibiotic treatment was given and 1 week later the patients were rechecked by scrotal ultrasonography. TB was performed monolaterally on the larger testis. When the volume of testes was the same, TB was always done on the right side. A 4–6 mm incision of the tunica albuginea (according to volume of testicle) was performed and a small testicular specimen was immediately transferred to Bouin’s solution. A second specimen of testicular parenchyma (50–200 mg, according to testicle size), was retrieved using gentle pressure on the testis. The testicular specimen was immediately collected in a tube filled with Quinn’s HTF medium and HEPES (Biocare Europe). Later, in the IVF laboratory, testicular parenchyma was transferred into Petri dishes with Quinn’s sperm washing medium (SAGE BioPharma, Bedminster, NJ, USA) and dissected into small pieces under a stereomicroscope using two coverslides (24 x 50 mm). The biopsy sample was collected in a sterile conic tube (Becton Dickinson Labware Europe, Le Pont De Claux, France) and centrifuged at 600/800 g for 10 min. To estimate the amount of retrieved spermatozoa, a scale of five degrees based on that described by Hauser et al. (1998)Go was adopted: 0 = no spermatozoa observed; 1 = a single spermatozoon occasionally observed after reviewing several microscopic fields (200x); 2 = a single spermatozoon observed in each microscopic field; 3 = a few spermatozoa in each field; 4 = many spermatozoa in each field.

After dilution with freezing medium (Irvine Scientific, Santa Ana, CA, USA), 0.25/0.50 ml sterile straws were loaded and frozen following a rapid two-phase protocol (Wolf and Patton, 1989Go).

TESE was considered successful even when a single spermatozoon was observed in testicular tissue. Nevertheless, for a more realistic classification, according to our experience in freezing–thawing spermatozoa, only >=2 degrees was considered suitable for more than one ICSI cycle with thawed sperm, since spermatozoa can be divided into multiple straws prior to cryopreservation, and each aliquot can potentially serve as a sperm source for a single ICSI attempt.

Diagnostic testicular biopsy was histopathologically classified according to the following patterns: (i) normal spermatogenesis; (ii) hypospermatogenesis; (iii) complete maturation arrest; and (iv) pure Sertoli cell-only syndrome (SCOS) (Tournaye et al., 1997Go). A quantitative evaluation of mature spermatids (mean number of completely elongated spermatids in at least 20 sections of tubules) was also performed (Silber et al., 1997Go). Referring to Silber’s studies, hypospermatogenesis was arbitrarily classified as severe or mild when <6 or 6–16 mature spermatids/seminiferous tubule cross-sections were observed, respectively. Consequently, normal (or substantially normal) spermatogenesis refers to the observation of a mean >16 mature spermatids/seminiferous tubule, in spite of the observation of areas of maturation arrest, SCOS or tubular sclerosis. Histological pictures of ‘incomplete maturation arrest’, ‘prevalent SCOS’ and ‘mixed anomalies’ were not considered, being included in ‘mild’ or ‘severe hypospermatogenesis’.

Statistics
Results are presented as mean ± SD. Data analyses were performed by unpaired t-test, Mann–Whitney U-test, Fisher’s exact test and {chi}2-test for evaluation of differences as appropriate for data type and distribution. The predictors of success (finding sperm for ICSI) were evaluated by performing univariate and multivariate logistic regression analysis using the statistical computer programme STATA6 (www.stata.com.). P < 0.05 was considered as statistically significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Data related to non-invasive diagnostics are reported in Table I. While mean FSH level in ex-cryptorchid patients was lower than the level observed in NOA patients (P = 0.04), no difference in orchidometry was observed. Scrotal ultrasonography revealed gross anomalies of the testicular parenchyma only in ex-cryptorchid men. No subclinical testicular cancer was observed in this patient population. Data related to suggestive signs of seminal duct obstruction, asymptomatic uroseminal infection or inflammation are reported in Table I. In two ex-cryptorchid men, congenital bilateral absence of the vas deferens was confirmed by cystic fibrosis gene mutation screening ({Delta}F-508 mutation in both cases) and by transrectal ultrasound (bilateral agenesis of the seminal vesicles). Referring to genetic screening, the prevalence of chromosomal abnormalities was 4.6% (5/107). All these cases referred to NOA patients [one case of Klinefelter’s syndrome; one case of Klinefelter’s mosaicism 47,XXY/46,XY; one 47XYY trisomy plus 13q14q Robertsonian translocation; and two Robertsonian translocations (13q14q and 14q22q)].


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Table I. Clinical data obtained from non-invasive diagnostics
 
Six cases of Y chromosome microdeletion were discovered in NOA patients. Five men had deletions in the AZFc region and one in AZFa region. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene were found in the two above-mentioned ex-cryptorchid men.

Data related to testicular histology are reported in Table II. An obstructive pattern (normal or mild hypospermatogenesis) resulted more frequent in ex-cryptorchid compared with NOA patients (P = 0.0004).


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Table II. Differences in the distribution of histological patterns in ex-cryptorchid and NOA populations
 
In 53 out of 107 patients (49.5%), TESE yielded a positive sperm retrieval. Detailed results are shown in Table III. No difference in age, testis volume and FSH level was observed between sperm-positive and sperm-negative groups.


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Table III. TESE outcome according to Hauser’sa scale in patients with previous history of cryptorchidism and in NOA patients without previous history of maldescended testes
 
No spermatozoa were detected after an extensive and meticulous search in 8/30 ex-cryptorchid patients and in 46/77 NOA patients (P = 0.0026). Conversely, a safe sperm extraction (grade 2–4) was reached in 13/30 ex-cryptorchid patients compared with 10/77 NOA patients (P = 0.0013). In ex-cryptorchid patients, no correlation was found between the age at bilateral orchidopexy and sperm retrieval after TESE.

Classical clinical variables, i.e. age, FSH level and volume of the larger testis, did not correlate significantly with the chance of retrieving testicular spermatozoa (see Table IV). Neither karyotype anomalies nor Y chromosome microdeletions correlate with TESE results, although the number of cases and the peculiarity of chromosomal anomalies found in this population of azoospermic men make these findings unreliable. Of five patients affected by karyotype anomalies, spermatozoa were retrieved only in one case (Robertsonian translocations 13q14q). In patients affected by deletions in the AZFc region (n = 5), sporadic spermatozoa were retrieved in only one patient with SY255 deletion. Sporadic spermatozoa were also extracted in the patient with deletion of AZFa region (SY81).


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Table IV. Correlation between the chance of retrieving testicular spermatozoa and clinical, non-invasive variables, evaluated by uni- and multivariate logistic analysis
 
Palpatory and/or ultrasonographic signs suggesting seminal duct obstruction were significantly correlated with TESE results in univariate analysis, but when considering multivariate analysis, this lost significance. In 14/22 men with associated obstructive signs grade 2–4 sperm retrieval was obtained. Sporadic spermatozoa were retrieved in two, and no spermatozoa in the remaining six patients.

A clinical history of a bilateral orchidopexy proved to be the most favourable variable for retrieving testicular spermatozoa, with an odds ratio (OR) resulting from multivariate analysis of 3.8 [95% confidence interval (CI) 1.41–10.21; P = 0.008]. The presence of seminal infection/inflammation in azoospermic men with secretive traits also proved to be a favourable factor in positive sperm retrieval (OR resulting from multivariate analysis of 3.6; 95% CI 1.12–11.57; P = 0.031).

In five out of six ex-cryptorchid patients who had sporadic spermatozoa in past semen analyses, spermatozoa were retrieved by TESE (grade 2–4). In six out seven NOA patients who had sporadic spermatozoa in past semen analyses, spermatozoa were retrieved by TESE (five grade 1, one grade 2).

Up to now, 63 ICSI cycles with thawed spermatozoa have been performed on 40 couples with sperm-positive retrieval (Table V). No differences in fertilization, implantation and clinical pregnancies were observed between the two groups.


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Table V. ICSI outcome using thawed spermatozoa in patients with previous history of cryptorchidism and in NOA patients without previous history of maldescended testes
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Accurate prediction of TESE has become essential since ICSI using testicular spermatozoa has been widely applied. While patients with obstructive patterns have a good prognosis, patients with clear secretive traits have a high prevalence of chromosomal abnormalities and a low probability for retrieval of spermatozoa from testicular tissue (nearly 50%) (Tournaye, 1999Go). Unfortunately, a clear obstructive/secretory pattern cannot be proven in some azoospermic men at the time of initial consultation. Histological visualization of spermatids provided the best predictive parameter of successful testicular sperm extraction in these patients (Ezeh et al., 1999Go); however, testicular histology is rarely available at the time of the patients’ initial consultation (Ezeh, 2000Go). Therefore, testicular volume, FSH level and clinical history remain the only inexpensive tools for the clinician counselling a couple with an azoospermic male.

Bilateral cryptorchidism is universally associated with a non-obstructive testicular dysfunction. Sharif (2000)Go includes cryptorchidism in testicular azoospermiae, such as other congenital (Klinefelter’s syndrome, Y deletion) or acquired (radiotherapy, chemotherapy, torsion, mumps orchitis) pathologies. Nieschlag et al. (2000)Go report that two-thirds of 163 patients with a history of maldescended testes suffer from SCOS or from maturation arrest; Glander et al. (2000)Go found that patients with a history of cryptorchidism show a significantly lower Johnsen score compared with the patients who had not had any testicular disease in the past, and they underline the negative effects of cryptorchidism on TESE results. Despite these data, our results suggest: (i) a high prevalence of obstructions (referring to histological pictures and TESE outcomes) in orchidopexed azoospermic men (~40%) compared with NOA patients without maldescended testes; and (ii) a low prevalence of chromosomal abnormalities and Y microdeletions in orchidopexed azoospermic men, recently confirmed by Kunej et al. (2003)Go.

Some paediatric and urological studies indicate that a high rate of obstructive patterns in ex-cryptorchid men is not surprising. The association between testicular maldescent and seminal duct anomalies (from rete testis to vas deferens) is a well-known fact. Prevalence of congenital epididymal or vasal anomalies is 36–43% in orchidopexed patients (Gill et al., 1989Go; Cicigoi and Bianchi, 1991Go; Mollaeian et al., 1994Go; D’Agostino et al., 1996Go; Nistal and Paniagua, 1997Go; Nistal et al., 2002Go). Biopsy of the testes with severe anomalies of ductal fusion shows preservation of germ cells in 69% and diminished germ cells in 31% (Gill et al., 1989Go). The prevalence and the severity of these anomalies appear to be strictly correlated to the level of undescended testis (Cicigoi and Bianchi, 1991Go) and with the degree of patency of the processus vaginalis (closed, partially closed or open) (Barthold and Redman, 1996Go).

Not to be underestimated is the risk of a iatrogenic obstruction, by transection of the vas deferens or by an excessive tension exerted on the spermatic cord (Adamsen and Borjesson, 1988Go; Redman, 2000Go). Therefore, these data support our TESE results; i.e. some form of obstruction is the cause or an associated cause of azoospermia in about half of orchidopexed men (those with normal spermatogenesis or mild hypospermatogenesis).

Secondary to these considerations, we wish to point out that the presence of silent uroseminal infection/inflammation has proved to be a positive prognostic factor in the retrieval of testicular spermatozoa. Cumulatively, in 14/19 azoospermic patients with secretive traits coupled with uroseminal infection/inflammation, TESE allowed a positive sperm retrieval. We can presume that whilst asymptomatic uroseminal infection-inflammation has little repercussion on sperm count in men with normal spermatogenesis, it can precipitate a severe oligozoospermia towards azoospermia in males with seriously damaged spermatogenesis, through an obstructive mechanism (Keck et al., 1998Go).

Based on the results of the present study we may conclude the following. (i) In the absence of a testicular histological examination, the clinical variable ‘bilateral orchidopexy’ should not represent a negative prognostic factor of TESE outcome in azoospermic patients. (ii) Azoospermiae found in orchidopexed men should not automatically be included in secretory (Ezeh et al., 1999Go) or in testicular azoospermiae (Sharif, 2000Go), being often associated with a congenital (or surgical) obstructive anomaly of the seminal ducts. The coexistence of defective spermatogenesis, i.e. from mild to severe hypospermatogenesis (justifying mild or severe oligozoospermia), with a congenital seminal duct anomaly (justifying the actual azoospermia), would suggest considering a new type of azoospermia, i.e. ‘mixed azoospermia’, as already hypothesized by Ezeh (2000)Go and Colpi et al. (2000)Go. Nevertheless, contrary to that affirmed by Ezeh (2000)Go, a mixed pattern would not appear to be particularly rare, and was applicable in 18/30 of our cryptorchid azoospermic patients with mild or severe hypospermatogenesis (~60%). (iii) An accurate evaluation of silent uroseminal infection/inflammation in presumed NOA patients seems advisable, since it has proved to be a positive prognostic factor in the retrieval of testicular spermatozoa in our patient population.


    Acknowledgements
 
The authors gratefully acknowledge the contribution of Dr Emanuela Morenghi for her assistance with the statistical analysis and Rosalind Roberts for reviewing the manuscript. The authors also thank Professor Giorgio Pardi for his suggestions.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on April 22, 2003; resubmitted on July 2, 2003; accepted on September 2, 2003.