1 Departments of Urology 2 Pathology 3 Obstetrics, Gynecology and Reproductive Sciences and 4 Physiology, University of California San Francisco School of Medicine, California, USA
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Abstract |
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Key words: azoospermia/histology/infertility/testis/vas deferens
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Introduction |
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It is generally assumed that azoospermia and infertility in men with CBAVD are due simply to obstruction and that sperm production is normal. On review of the literature, however, there is evidence that this assumption may not be valid. Okada et al. (1999) found that spermatogenesis `was not significantly impaired' in their cohort of 10 men with CBAVD who had testis biopsies. Three men, however, had evidence of hypospermatogenesis suggested by a low Johnsen's score. Similarly, Goldstein and Schlossberg reported normal spermatogenesis in seven men and slight hypospermatogenesis in two men with CBAVD who had testis biopsies (Goldstein and Schlossberg, 1988).
The advent of assisted reproductive techniques has allowed paternity in cases of obstructive azoospermia, including CBAVD. Both microsurgical epididymal sperm aspiration (MESA) and testicular sperm extraction (TESE) are ways to obtain sperm for intracytoplasmic sperm injection (ICSI) in men with CBAVD (Schlegel et al., 1995; Okada et al., 1999
). Since reproductive technology is costly, both financially and emotionally, the true reproductive potential of patients with CBAVD deserves further investigation in the light of current evidence and assumptions. We evaluated spermatogenesis in patients with CBAVD to determine whether or not it is uniformly normal.
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Materials and methods |
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Genetic analysis
All patients (n = 33) were offered CF mutation and variant analysis from blood to evaluate the genetic basis for CBAVD; 28 men elected to undergo such testing. Men with evidence of impaired spermatogenesis on biopsy or physical examination were offered karyotype and Y chromosome microdeletion analysis.
For patients of Caucasian and Hispanic ancestry, CFTR mutation analysis was performed on blood by DNA extraction, polymerase chain reaction (PCR) amplification, and hybridization to allele-specific oligonucleotides for the 31 most common mutations, yielding a 90% and 57% detection rate respectively in these ethnic groups. For patients of Asian ancestry, CFTR mutation analysis was performed by DNA extraction from blood followed by several PCR-based assays, including single-stranded conformation polymorphism. This process tested for more than 48 mutations, with a reported 6080% detection rate. For all patients seen in the past 9 months, CFTR variable 5T, 7T, and 9T repeats of intron 8 were analysed on leukocyte DNA, amplified by PCR, and hybridized to allele-specific oligonucleotides in the reverse dot blot format.
For karyotype analysis, peripheral blood was cultured and chromosomes were analysed using the GTW banding method. At least 20 cells were examined from each sample.
Y chromosome microdeletion analysis was performed on DNA extracted from peripheral lymphocytes by PCR analysis. Twenty-four carefully selected primers blanketed both the long and short arms of the Y chromosome, and a marker was not recorded as absent until three successive attempts to PCR amplify the locus yielded negative results.
Testis biopsy technique and interpretation
Testis biopsies were obtained by the open `window' technique under local anaesthesia (Coburn and Wheeler, 1991). Testis biopsies were stained with haematoxylin and eosin to visualize histological details and classified according to Levin: normal spermatogenesis, germ-cell hypoplasia or hypospermatogenesis, early or late maturation arrest, germ-cell aplasia (i.e. Sertoli cell only), and tubular sclerosis (Levin, 1979
). All testis biopsies were interpreted by a single pathologist (I.C.).
FNA technique and interpretation
The FNA procedure was performed as previously described (Turek et al., 1997). Briefly, the scrotal skin was cleaned and the spermatic cord infiltrated with local anaesthesia. The scrotal skin was stretched taut over the testis. Planned aspiration sites were marked on the scrotal skin overlying the testis and placed ~57mm apart. FNA was performed with a sharp-bevelled, 23 gauge, 1 inch fine needle (Becton-Dickinson Co., Franklin Lakes, NJ, USA) using the established suction-cutting technique (Ljung, 1992
). 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 58 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, smeared, and fixed in 95% ethyl alcohol. Pressure was applied to each aspiration site for haemostasis. A routine Papanicolaou stain was performed on the smear.
Each stained FNA cytologic smear was interpreted by an experienced cytopathologist for (i) the presence or absence of mature sperm with tails, and (ii) specimen adequacy, as previously reported (Turek et al., 1997). An adequate and informative FNA specimen was defined as that which contains at least 100 clusters of 20 or more testis cells or at least 2000 well-dispersed testis cells.
Microscopic epididymal sperm aspiration
MESA was performed as previously described (Nudell et al., 1998). Briefly, local anaesthesia was used to obtain a spermatic cord block and to anaesthetize the scrotal skin. The testis was held so that the scrotal skin was stretched tightly over the testis. A 1 cm transverse incision was made in the upper, lateral scrotum and the tunica vaginalis space entered. A self-retaining eyelid retractor was placed in the incision to create a `window' into the tunica vaginalis space. Suitable dilated epididymal tubules were identified under x25 magnification. Using microsurgical technique, the epididymal tunic was incised and an individual tubule isolated. The epididymal tubule was entered with microscissors. Fluid and spermatozoa were aspirated with a 24-gauge angiocath-catheter attached to a 1.0 ml syringe prefilled with Earle's medium supplemented with 4 mmol/l sodium bicarbonate, 21 mmol/l HEPES, 0.47 mmol/l pyruvate, and 10% v/v synthetic serum substitute. At each aspiration site, 10 µl of extracted fluid was examined under x400 bright-field microscopy for the presence of motile spermatozoa.
After surgical retrieval, sperm quality was formally analysed by an andrologist in the IVF laboratory. Aspirate volume, sperm concentration, percentage motility and forward progression were assessed according to established methods (WHO, 1992). The total motile sperm count was calculated using the formula: (volume) x (sperm concentration) x (motile fraction).
Testicular sperm extraction
TESE was performed as described by Turek et al. (1999). Briefly, anaesthesia was achieved using spermatic cord and scrotal skin blocks and a 1 cm incision was made in the scrotal skin, similar to a routine `window' testis biopsy. Four-power optical magnification was used for the procedure. A single, small biopsy was taken (~75 mg) and immersed in 1.0 ml of Earle's medium supplemented with 4 mmol/l sodium bicarbonate, 21 mmol/l HEPES, 0.47 mmol/l pyruvate, and 10% v/v synthetic serum substitute at 37°C. The specimen was macerated lightly with sterile scalpel blades, transferred to the IVF laboratory, and examined for the presence or absence of mature spermatozoa. Sperm quality was analysed with respect to volume of tissue recovered, sperm concentration, and percentage motility.
Definitions of normal spermatogenesis
Information from each diagnostic and therapeutic procedure was evaluated in an attempt to quantify spermatogenesis. Based on previous experience with FNA, MESA, and TESE, we established definitions of normal spermatogenesis for each diagnostic and therapeutic procedure. These definitions are listed in Table I and are based simply on findings and clinical experience from men who were considered to have `normal' spermatogenesis (Silber and Rodriguez-Rigau, 1981
; Turek et al., 1997
; Nudell et al., 1998
).
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Results |
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Discussion |
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There are studies which suggest that spermatogenesis is normal in men with CBAVD. In separate reports, Goldstein and Schlossberg (1988) and Okada et al. (1999) performed testis biopsies in 19 men with CBAVD and observed the presence of spermatogenesis in all men. Schlegel et al. (1995) retrieved a mean of 79x106 spermatozoa/patient, with 10% motility, from the epididymis in men with CBAVD. In this study, there were no differences in fertilization or pregnancy rates between men with or without detectable CFTR mutations. Thus, the presence of CFTR mutations did not adversely affect sperm function with ICSI. Taken together, this information suggests that spermatogenesis is normal in men with CBAVD.
Several observations, however, point to the potential for abnormalities in spermatogenesis in CBAVD patients. In patients studied by Okada et al. (1999), there was histological evidence of hypospermatogenesis in 30% of CBAVD men. Further reading of the study by Goldstein and Schlossberg (1988) reveals that `slight hypospermatogenesis' was observed in 22% of patients with testis biopsies. Our results provide further confirmatory evidence that abnormalities in spermatogenesis can exist in men with CBAVD. In this cohort of 33 men, 12% demonstrated abnormal sperm production. On clinical evaluation, our data demonstrate that patients with abnormal spermatogenesis are more likely to have elevated FSH levels and smaller testicles than their counterparts with normal spermatogenesis. This clinical profile is similar to that found in the general population of infertile men with testis failure. We suggest that men with CBAVD and these two risk factors be considered for further evaluation of sperm production.
Several aetiologies may account for the observed defects in spermatogenesis. These include effects of CFTR gene defects, genetic and other testicular conditions unrelated to CF, and changes in spermatogenesis resulting from chronic obstruction.
Although there is evidence to suggest that alterations in the CFTR gene may adversely impact spermatogenesis, our data seem to indicate that the impaired spermatogenesis in men with CBAVD is not related to CFTR mutations or splice variants. In our cohort, the majority of men with classic CFTR mutations demonstrated normal spermatogenesis. In addition, the 5T splice variant was not found more commonly in the subset with abnormal spermatogenesis.
Two patients demonstrated genetic conditions unrelated to CFTR and CBAVD (AZFb microdeletion and chromosomal inversion) which likely account for impaired spermatogenesis. This is not an unexpected finding given the relatively high incidence of Y chromosome microdeletions and karyotypic abnormalities among infertile men in general. Men with CBAVD are also susceptible to testicular causes of azoospermia, including varicocele, and this should be kept in mind during their evaluation.
Another mechanism of decreased spermatogenesis may be related to the presence of obstruction, as suggested by Jarow et al. (1985). Multiple studies in animals and humans have evaluated changes in testicular morphology after vasectomy. Some of these alterations include autoimmune reactions, thickening of the tunica propria of the seminiferous tubular wall, and interstitial fibrosis. Jarow et al. quantified pathological changes in human testes after vasectomy and observed similar changes and also reduced numbers of Sertoli cells and spermatids. Chronic obstruction in CBAVD could mimic the situation after vasectomy, and thus impaired spermatogenesis in CBAVD may, in part, result simply from obstruction. However, we did not find a statistically significant difference in age between men with normal and abnormal spermatogenesis.
In evaluation of men with CBAVD, especially those considering assisted reproduction, the potential for impaired sperm production exists. While vasal obstruction alone or CFTR alterations may result in abnormal spermatogenesis, we found reasons unrelated to CF and CBAVD for abnormal sperm production in these men. This indicates that (i) we should not assume men with obstruction secondary to CBAVD have uniformly normal spermatogenesis and (ii) men with CBAVD are susceptible to genetic and testicular causes of impaired spermatogenesis, similar to unobstructed infertile men. Patients should be informed of the risks of MESA/TESE failure, and the clinical suspicion of testis failure should prompt further evaluation. We found a significant increase in serum FSH concentration and decrease in testicular volumes in CBAVD men with impaired spermatogenesis. Testis biopsy with careful histological evaluation can provide important information regarding options for ICSI. In addition, further genetic counselling and testing should be offered to provide more complete information on transmissible genetic conditions that may underlie abnormal spermatogenesis in these men.
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Notes |
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References |
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Submitted on August 21, 2000; accepted on December 7, 2000.