1 Centre for Reproductive Medicine and 2 Department of Clinical Immunology, Sahlgrenska University Hospital, Göteborg University, Göteborg, Sweden
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Abstract |
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Key words: antisperm antibodies/azoospermia/male infertility/testicular sperm aspiration/ultrasonography
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Introduction |
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To optimize the possibility of achieving successful sperm recovery, multiple (Hauser et al., 1998) and bilateral (Plas et al., 1999
) testicular sampling is recommended. Several reports have recently been published using different methods for testicular sperm retrieval in men with NOA. Some groups have emphasized that TESE is more superior technique (Friedler et al., 1997
; Ezeh et al., 1998
), while others have reported TESA to be almost as effective as the more invasive TESE procedure (Lewin et al., 1999
; Westlander et al., 1999
). Microsurgical TESE is a novel approach to the identification of focal areas with sperm production and in two prospective studies was considered more effective and less traumatic than conventional TESE (Schlegel, 1999
; Amer et al., 2000
). Multiple gun needle biopsy is a promising new sperm recovery technique described in men with both obstructive (Harrington et al., 1996
; Steele et al., 2000
) and non-obstructive (Tuuri et al., 1999
) azoospermia.
There are only a few published reports where the physiological consequences of different sperm recovery techniques have been studied. The procedures are often repeated and their consequences for the testis are still unknown. Open biopsies of the testis increase the risk of vascular injuries and inflammatory changes. Serial sonography after TESE has shown a high percentage of persistent intratesticular abnormalities (Schlegel and Su, 1997; Ron-El et al., 1998
). The consequences of the less invasive testicular puncture techniques have so far not been studied.
The correlation between antisperm antibodies (ASA) and infertility is controversial. The prevalence of ASA is difficult to estimate due to variable specimen preparation, different testing methods and subjective test interpretation (Mazumdar and Levine, 1998). Several aetiologies of ASA formation have been described, such as cystic fibrosis (Vasquez-Lewin et al., 1994), vasectomy (Jarow et al., 1994
), childhood inguinal herniorrhaphy (Matsuda et al., 1992
), varicocele (Gilbert et al., 1989
) and genital tract infection (Mazumdar and Levine, 1998
).
The aim of this prospective study was to investigate the physiological consequences of TESA, using puncture with a 19 gauge needle with suction, evaluated by serial ultrasonography, consecutive analyses of serum FSH and testosterone concentrations and formation of ASA in serum.
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Materials and methods |
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A sequential unselected series of 35 men with azoospermia underwent attempted TESA in conjunction with a diagnostic sperm retrieval procedure. The mean (± SD) age of the men was 33.3 (± 5.52) years (range 2648). Azoospermia had earlier been confirmed by at least two semen analyses. A detailed history and physical examination were performed on all patients. Patient characteristics are described in Table I.
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Sonography and Doppler imaging
The ultrasound examinations were performed by one gynaecologist (G.W.) using an ATL HDI 5000 machine (ATL Ultrasound, Bothell, WA, USA) equipped with a linear broadband transducer operating at the range 512 MHz in both two-dimension and colour. Testicular sonographic echogenicity was evaluated using a semi-qualitative score (Westlander et al., 2001a). This is a modification of a score used by Lenz and co-workers (Lenz et al., 1993
) (Figure 1
). Colour Doppler imaging was used to identify vessels within the testicular parenchyma. To optimize the imaging, all scans were performed in multiple orientations. Testicular parenchyma with homogeneous distribution of vessels was considered normal and parenchyma with either a poor distribution with a low amount of vessels or focal devascularized regions with hypo- and/or hyperechoic structures was considered abnormal. Microcalcifications were diagnosed as hyperechoic areas and, if there were no signs of cysts (hypoechoic areas), patients with these findings were placed in category 3.
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Determination of anti-sperm antibodies
The concentration of ASA in serum was analysed using a pool of test spermatozoa collected from five different donors and stored in aliquots in liquid nitrogen until use. Before use, the spermatozoa were thawed in a 37°C water bath and washed twice in phosphate-buffered saline (PBS) containing 5 mmol/l EDTA (PBS/EDTA). The serum samples were diluted 1:5 and incubated with 35x105 spermatozoa in 100 µl PBS on ice for 30 min. Thereafter, the spermatozoa were washed twice in PBS/EDTA and then fluorescein isothiocyanate (FITC)-labelled rabbit (Fab) 2 antibodies specific for human immunoglobulin (Ig)G, IgA or IgM (Dako, Glostrup, Denmark) were added to each sample at 1:20 dilution in 100 µl PBS. After an additional incubation for 30 min on ice, the spermatozoa were washed once in PBS/EDTA and then the cells were rinsed once and diluted to an appropriate concentration in fluorescence-activated cell sorter (FACS) flow buffer (Becton Dickinson, San Jose, CA, USA). Immediately before analysis, 5 µl propidium iodide (PI) at 5 mg/ml was added to each sample and the amount of binding of ASA was determined by FACS analysis (FACScan, Becton Dickinson). The amount of bound ASA in different Ig classes was detected by gating on spermatozoa that were negative for PI, a marker for viable cells. An objective measure of the concentration of bound ASA was achieved by setting gates on PI-negative spermatozoa, which were then analysed for the expression concentration of ASA, as reflected in the amount of fluorescence and depicted as histograms. The detection threshold for a positive result was set by using one negative and two positive serum samples from the National Institute for Biological Standards (Holly Hill, Hampstead, UK; 69/65, 69/82 and 280) for assessing the presence of ASA. Thereafter, gates for positive serum samples were set and the mean fluorescence intensity (MFI) in the gated population was used as an indirect measure of the concentration of ASA in each individual sample. Values were given as mean MFI ± SD for three determinations. A MFI value >30 was considered positive when serum samples were analysed at 1:5 dilution.
TESA
Alfentanil 0.5 mg (Rapifen®, Jansen-Cilag, Beerse, Belgium) was given i.v. followed by infiltration of 68 ml lidocain/bupivacain (equal volumes; Xylocain®/Marcain®, Astra, Södertälje, Sweden) around the spermatic cord. Under sterile conditions, a 19 gauge butterfly needle was passed through the scrotal skin. Suction was applied with a 20 ml syringe and the negative pressure was maintained by clamping the distal end of the needle tubing with two pairs of forceps. The needle was pushed 46 times in different directions with quick thrusting movements into the testicular tissue. The needle was then slowly removed from the testis and the scrotal skin while the back pressure was maintained by the clamped tubing. The assistant used two pairs of fine tweezers to pick up the small tubules recovered by the needle. The clamps were then removed and the needle was flushed with culture medium and its content expelled into a sterile tube containing culture medium (IVF-50, Scandinavian IVF Science, Göteborg, Sweden). Three to five punctures (mean 3.92 ± 0.53) were performed in different regions at the ventral surface of each testis. The testicular tissue was dissected, incubated in culture medium and examined 1, 4 and 24 h after puncture. If spermatozoa were found, cryopreservation was possible for subsequent ICSI cycles.
Follow-up
Three months after TESA, physical examination including estimation of testicular volume was repeated. Sonographic examination with Doppler imaging was performed and if the examination revealed any abnormal extra- or intratesticular findings, the patients were requested to come for repeat ultrasonography 6 months after TESA. All the testicular parenchyma was evaluated and images showing any hyper- or hypoechogenic structures and the distribution of intratesticular vessels were saved on a magneto optical disc. Any per- or post-operative complication was documented and the patients were questioned about any physical subjective discomfort.
All the images on the magneto optical disc were later evaluated blindly by two investigators (E.E. and S.G.). Intratesticular echogenicity was estimated, using a semi-qualitative score (Figure 1). In cases where the investigators estimated images differently, they discussed the images and came to an agreement.
Serum levels of FSH and testosterone and possible formation of ASA were analysed. The same analyses were also repeated 6 months after TESA.
Statistical analysis
Descriptive statistics are given as mean, SD, median and range. Paired comparisons were performed for FSH, testosterone and testicular volume using the non-parametric permutation test. All comparisons were performed on the individual level. A P-value < 0.05 was considered significant.
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Results |
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In four cases (11%) the discomfort was more intense. One of these men (case 17) had a medical consultation on day 7 after retrieval, when a hypoechoic and homogeneous cystic lesion with a diameter of 10 mm was diagnosed by ultrasound. The discomfort had disappeared after 7 days. Another man experienced intense discomfort for 67 days with bilaterally swollen testes, but abstained from medical consultation. Ultrasound scanning 3 months after TESA revealed no intratesticular abnormality. Both testes were unchanged in volume. The last two cases abstained from medical consultation and claimed intense subjective discomfort for 7 days, but denied any swelling of the scrotal region.
Ultrasound scanning revealed unchanged intratesticular echogenicity (same category) in 50 out of 61 examined testes (82%) 3 months post-surgery (Table II). In nine testes the echogenicity was considered more abnormal (higher category). In five of these cases the testicular texture in general had become more heterogeneous and in four cases focal lesions were observed. In two testes, improved echogenicity (lower category) was found compared with before surgery. All changes were seen unilaterally. In no case did post-operative Doppler imaging show a reduced amount of intratesticular vessels compared with the pre-operative scanning.
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Serum FSH and testosterone did not differ when comparing pre-operative with post-operative levels 3 and 6 months after TESA. There were no differences in testicular volumes before and 3 months after TESA when calculating per patient (Table III).
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Discussion |
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On the other hand, several studies have been published where the consequences of testicular biopsies have been evaluated. Persistent devascularized focal lesions were detected by ultrasonography in nine out of 14 men (64%) (Schlegel and Su, 1997) and 11 out of 14 men (79%) (Ron-El et al., 1998
) respectively, 6 months after the TESE procedure. Another published study reported a lower risk of complications with microdissection TESE compared with conventional TESE (Amer et al., 2000
).
Percutaneous needle biopsy is easier to perform and the intratesticular ultrasound appearance after such biopsies has been studied. In one report, scrotal ultrasound revealed sonographic evidence of intratesticular bleeding in two out of 58 needle biopsies (3.4%) 3 months after the procedure, and 10 out of 34 open biopsies (29%) 1 month after the procedure (Harrington et al., 1996). The lesions found after needle biopsy were all resolved after 6 months but all lesions found after open biopsy persisted for >6 months. The incidence of post-operative intratesticular lesions detected by ultrasound after needle biopsy in that study was in the same range as our TESA results (6.6%). However, Harrington and colleagues only performed one needle biopsy on each testis, while in the present study 35 needle aspirations were performed.
Repeated aspiration procedures have previously shown satisfactory recovery of sperm and fertilization after PESA (Rosenlund et al., 1998) and after TESA (Westlander et al., 2001b
).
In the present paper, the physiological consequences of TESA were studied. In four out of 35 men, ultrasonography revealed intratesticular lesions consistent with haematomas and inflammation at TESA sites or intratesticular bleeding from the TESA procedures. All discovered lesions resolved spontaneously. It is known that the normal testis has an almost regular echo pattern (Lenz et al., 1993). In 26 men (52 out of 61 operated testes) there was no tendency towards deterioration in testicular echogenicity 3 months after surgery. In five men where no lesions were found post-operatively, the echogenicity of the testicular parenchyma was still considered less normal than before the procedure (Table II
). Further, in two other cases the echogenicity of the testicular texture had improved after recovery. The reason for these changes is not clear. The number of patients with changed echogenicity was small. The evaluation of the ultrasound images was, of course, subjective and there is always a risk of methodological unreliability. However, changed testicular texture as a physiological consequence of sperm retrieval is possible.
There were no significant differences between pre- and post-operative levels of serum FSH and testosterone.
No post-operative formation of ASA in serum was seen in any of the 35 patients. However, two patients had high IgG titres at 3 and 6 months after the procedure, but similar ASA levels were also present pre-operatively. In addition, three men showed seroconversion with IgG or IgM at 3 months post-surgery, declining at 6 months, but the concentrations were only borderline ASA-positive.
An earlier study (Harrington et al., 1996) reported no new or increased ASA titres in serum or seminal fluid in patients with azoospermia or severe oligozoospermia who had undergone percutaneous testis biopsy with an 18 gauge biopsy needle or open testicular biopsy 6 months earlier. Seven percent out of 31 men in the percutaneous and 5% out of 20 men in the open testis biopsy group had ASA bound to spermatozoa or in serum pre-operatively, which is comparable with the current results, where ASA in serum was found in two out of 35 men (5.7%) pre-operatively.
The method used in the current study for analysis of ASA is very sensitive. Detection of ASA with the FACS technique allows objective assessment of the concentration of ASA, as reflected in the amount of specific antibody that binds to the spermatozoa. Thus, the MFI values can be compared between different samples from different individuals as well as between the three time-points for each single individual. Although not positive by definition (i.e. MFI <30), it was noted that the MFI values for three individuals showed seroconversion from day 0 to 3 months for IgG or IgM ASA.
In four out of five cases where ASA FACS analysis was able to detect positive findings, sperm recovery was successful. Three of these men suffered from obstructive azoospermia and one from NOA. It has been suggested that active spermatogenesis is a prerequisite for the development of ASA in men (Mazumdar and Levine, 1998). However, in one of the men in the current study, sperm recovery was unsuccessful with both TESA and TESE. After a subsequent TESE procedure, histopathology revealed Sertoli cell-only syndrome. A possible explanation could be focal areas of spermatogenesis which were not discovered or spermatogenesis earlier in life.
Despite the fact that the time-course of ASA development is unclear, we chose not to follow the patients longer than 6 months after the procedure. Data from a rat model suggest that IgM ASA develop within 2 weeks after vasectomy and subsequently diminish over 48 weeks, followed by increasing titres of ASA IgG between 812 weeks (Flickinger et al., 1994). The clinical consequences of ASA in infertile couples are still not clear and remain controversial. It has been argued that there is sufficient evidence for a role of ASA in relation to spontaneous conception in selected couples with unexplained infertility (Mazumdar and Levine, 1998
).
Negative results have been reported from couples with the presence of ASA in both women and men. Lahteenmaki et al. reported lower fertilization rates, but similar pregnancy rates, in IVF cycles with high titres of sperm-bound ASA (Lahteenmaki et al., 1993). However, no negative effects on fertilization rates have been reported after ICSI, which is strongly recommended in severe male immune infertility with >80% antibody-bound spermatozoa (Lahteenmaki et al., 1995; Nagy et al., 1995
; Lundin and Hamberger, 1996
). A published study by Check et al. demonstrated comparable fertilization, pregnancy, implantation and miscarriage rates in female partners of males with and without sperm auto-antibodies (Check et al., 2000
). All of these studies included ICSI with ejaculated spermatozoa. Men with azoospermia are dependent on ICSI with surgically recovered spermatozoa. Whether formation of ASA in serum negatively affects the fertilization capacity of testicular spermatozoa is not known.
An accepted hypothesis for ASA formation is breaching of the bloodtestis barrier. If this hypothesis is true, development of ASA may occur after TESA as well as after other surgical techniques used for sperm retrieval.
In summary, intratesticular lesions diagnosed as resolving post-operative haematomas were found by ultrasound in 11% of the patients and in 7% of aspirated testes 3 months after TESA. The subjective discomfort was mostly mild and none of the lesions were permanent. Serum FSH and testosterone levels did not change after the procedure. There were three patients with borderline ASA formation but none were classified as ASA-positive. Testicular sperm aspiration seems to be a safe method for sperm retrieval with minimal physiological consequences in men with obstructive and non-obstructive azoospermia.
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Acknowledgements |
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Notes |
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References |
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Submitted on March 5, 2001; accepted on September 12, 2001.