Induction of spermatogenesis in azoospermic men after varicocelectomy repair: an update

Induction of spermatogenesis in azoospermic men after varicocelectomy repair: an update Fábio Firmbach Pasqualotto, M.D., Ph.D., Bernardo Passos Sobreiro, M.D., Jorge Hallak, M.D., Ph.D., Eleonora Bedin Pasqualotto, M.D., Ph.D., and Antônio Marmo Lucon, M.D., Ph.D. Divisão de Clínica Urológica, Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, São Paulo, and Universidade de Caxias do Sul, Caxias do Sul, Brazil Objective: To assess the treatment outcome after varicocele repair in azoospermic men and to correlate this outcome with the testicular histology patterns. Design: Prospective study. Setting: Academic medical centers. Patient(s): Medical records of 27 azoospermic men, who underwent testis biopsy and microsurgical repair of clinical varicocele between July 1999 and May 2004, were reviewed. Intervention(s): Twenty-seven azoospermic men underwent testis biopsy and microsurgical repair of clinical varicocele. All patients had at least two semen analyses showing azoospermia taken before the surgery and two semen analyses postoperatively. Main Outcome Measure(s): Semen analysis after varicocelectomy. Result(s): Hypospermatogenesis was identified in 9, maturation arrest in 8, and germ cell aplasia in 10 men. Induction of spermatogenesis was achieved in nine men (33.3%). Of these, four had germ cell aplasia, three had maturation arrest, and two had hypospermatogenesis. The improvement in sperm concentration and motility ranged from 1.2 10 6 /ml to 8.9 10 6 /ml, and from 24% to 75.7%, respectively. Of these nine patients with improvement in semen quality, five relapsed into azoospermia 6 months after the recovery of spermatogenesis (four germ cell aplasia and one maturation arrest). One patient with maturation arrest established pregnancy. Conclusion(s): Azoospermic patients may have an improvement in semen quality after varicocelectomy. Semen samples may be cryopreserved after an initial improvement after varicocelectomy. (Fertil Steril 2006;85:635 9. 2006 by American Society for Reproductive Medicine.) Key Words: Varicocele, azoospermia, semen, testis, cryopreservation Although the incidence of clinical varicocele in men in the general population is roughly 15%, it has been implicated as a factor responsible for infertility in as many as one-third of the infertile population (1 3). With recent advances in diagnostic techniques and widespread application of scrotal ultrasonography and color Doppler imaging, varicoceles are being reported in up to 91% of subfertile cases, most of whom were previously regarded as having idiopathic etiology (4, 5). A number of theories have been proposed to explain the observed pathophysiology of varicoceles. Semen quality uniformly declines in animals with induced varicoceles, even when only a left varicocele is produced (6). The reduction in scrotal temperature after varicocele ligation supports a causative role of increased temperature on the infertility produced by the varicocele (7). It has been hypothesized that varicoceles cause hypoxia, which may play a role in altering spermatogenesis in the varicocele patient. Infertile men with Received April 19, 2005; revised and accepted August 23, 2005. Reprint requests: Fabio Firmbach Pasqualotto, M.D, Ph.D., Pinheiro Machado, 2569, sl 23/24, Bairro São Pelegrino, Caxias do Sul, RS, Brazil, CEP 95020172 (FAX: 55 54 3215 1695; E-mail: Fabio@ conception-rs.combr). varicocele have elevated levels of spermatozoal reactive oxygen species (8, 9). The effects of the varicocele vary but may often result in a generalized impairment of sperm production, characterized by abnormal semen quality and the fertilizing capacity of the haploid male gamete (10). Although the true pathogenesis of the varicocele remains enigmatic, gross testicular alterations associated with varicocele are well documented and the fact that both oligoasthenoazoospermia and azoospermia indicate bilateral testicular dysfunction is in agreement with recent reports that varicocele is mainly a bilateral disease (11, 12). Induction of spermatogenesis in 7 of 15 azoospermic men (46.6%) after varicocelectomy (13) was in agreement with other investigators who found that varicocele repair in men with azoospermia and severe oligoasthenozoospermia may result in induction or enhancement of spermatogenesis in 40% 60% of the patients (14 16). On the other hand, the initial benefit of varicocele repair in azoospermic patients may be limited, with only 9.6% of men after varicocele repair having enough viable sperm in the ejaculate to avoid a testicular sperm extraction (TESE) procedure (17). Because of this discrepancy, the purpose of our study was to reassess the improvement in semen quality and pregnancy 0015-0282/06/$32.00 Fertility and Sterility Vol. 85, No. 3, March 2006 doi:10.1016/j.fertnstert.2005.08.043 Copyright 2006 American Society for Reproductive Medicine, Published by Elsevier Inc. 635

outcome after varicocelectomy in men with azoospermia. Increasing the numbers from 15 to 27 men, we also sought to correlate the testicular histology patterns of a group of azoospermic men with varicocele with the treatment outcome after varicocele repair. MATERIALS AND METHODS This study was approved by the University of São Paulo and University of Caxias do Sul review board and the patients involved granted their informed consent. In a prospective study, 27 azoospermic men with clinical varicocele underwent microsurgical varicocele repair between July 1999 and May 2004. All of them had primary infertility. The minimum duration of infertility required was defined as a failure to establish a pregnancy within 1 year with unprotected intercourse. A basic infertility evaluation including a detailed history and a complete physical examination was undertaken. Only patients with varicocele grade II III identified on physical examination were included. Patients who were taking antioxidants like vitamin C and E were excluded from the study. The mean age of the wife at presentation was 30.2 4.8 years (range 23 39 years). All women were normal based on history, hormonal levels, and hysterosalpingogram. Testicular size was evaluated in all patients with a caliper or by scrotal ultrasound. Testicular atrophy/hypotrophy ( 10 ml) was bilateral in six patients (22.2%) and unilateral in five (18.51%). The mean ( SD) right testes was 21.3 4.6 ml and left testes was 20.23 4.8 ml. Mean preoperative hormone levels were FSH, 17 12.4 miu/ml (range 8.5 38 miu/ml); LH, 12.5 9 miu/ml (range 6 43 miu/ml); and T 412 145 ng/dl (range 252 579 ng/dl). Karyotype and Y microdelection were available only in the last 12 patients operated, showing no evidence of abnormalities. All patients had at least two semen analyses showing azoospermia obtained by masturbation after 2 5 days of abstinence before the surgery. Samples were centrifuged at 600 g for 10 minutes and no sperm were detected in the pellet, confirming the diagnosis of azoospermia. Patients with pyospermia were treated before varicocele repair. Repair was bilateral in 15 and unilateral in 12 patients using a subinguinal approach and a microsurgical technique. Using the microsurgical approach, we ligate the pampiniform plexus, leaving intact the cremasterium plexus as well as the gubernaculum veins. Each patient underwent open diagnostic testis biopsy at the same time as the varicocele repair under general anesthesia. Biopsies were performed on both testes, irrespective of which appeared healthier whether by size or shape. Two new semen analyses were performed on each patient at 6 and 12 months postoperatively. Sperm concentration and motility were evaluated according to the World Health Organization criteria and sperm morphology according to Tygerberg s strict criteria. Samples were centrifuged at 600 g for 10 minutes and if no sperm were detected in the pellet, a diagnosis of azoospermia was confirmed. The biopsy results, postoperative semen analysis, and the correlation between the induction of spermatogenesis and the testis biopsy were studied. We also evaluated the pregnancy outcome after varicocele repair. RESULTS Germ cell aplasia was identified in 10, hypospermatogenesis in 9, and early maturation arrest (arrest at the primary spermatocyte stage) in 8 of the men (Table 1). Induction of spermatogenesis was achieved in nine of them (33.3%), six with bilateral (40%, 6/15) and three with unilateral varicoceles (25%, 3/12). Of these nine men, four had germ cell aplasia, three maturation arrest, and two hypospermatogenesis. After the varicocelectomy in men with germ cell aplasia, the mean SD sperm concentration was 1.6 2.59 10 6 /ml, the sperm motility (grades A B) was 18.8% 26.9%, and normal sperm morphology was 1.7% 2.3%. In men with maturation arrest, sperm concentration was 1.6 3.1 10 6 /ml, the sperm motility was 11.3% 16%, and the normal sperm morphology was 1.5% 2.3% postoperatively. Furthermore, in patients with hypospermatogenesis, the mean SD sperm concentration was 0.87% 1.74%, sperm motility was 6.7% 13.3%, and normal sperm morphology was 0.78% 1.7% after the surgery. The highest sperm concentration, motility, and morphology between the two semen analyses obtained after the surgical procedure are depicted in Table 1. Thirty-three percent (2/6) of the patients with bilateral testicular atrophy and high FSH levels, 20% (1/5) of the patients with unilateral atrophy with high FSH levels, and 37.5% (6/16) of the patients with normal testicular size and FSH levels had sperm in the semen. Of the nine patients with improved semen quality, five relapsed into azoospermia 6 months after the recovery of spermatogenesis (four germ cell aplasia and one maturation arrest). Of these 5 patients who relapsed, 3 underwent a bilateral varicocelectomy and 2, a unilateral procedure. The only patient who established a pregnancy had a testicular biopsy showing maturation arrest (Table 1). DISCUSSION The first study on the importance of varicocelectomy to male infertility was published in 1952 by Tulloch (18), who reported spontaneous pregnancy after varicocele repair in an azoospermic man. Since that time, varicocelectomy has become the most commonly performed surgery for male factor infertility. However, only in the past 10 years, some studies have shown that nonobstructive azoospermic patients with 636 Pasqualotto et al. Azoospermic men after varicocelectomy Vol. 85, No. 3, March 2006

Fertility and Sterility TABLE 1 Histology pattern of the testis in azoospermic man with varicocele and semen analysis (ejaculate specimen) after varicocelectomy. Patient Testis biopsy Varicocelectomy Concentration ( 10 6 /ml) Motility (%) Morphology Tygerberg (%) Pregnancy Azoospermia 1 Hypospermatogenesis Bilateral Zero Zero Zero No Yes 2 Hypospermatogenesis Unilateral Zero Zero Zero No Yes 3 Hypospermatogenesis Bilateral Zero Zero Zero No Yes 4 Hypospermatogenesis Bilateral Zero Zero Zero No Yes 5 Hypospermatogenesis Unilateral Zero Zero Zero No Yes 6 Hypospermatogenesis Unilateral Zero Zero Zero No Yes 7 Hypospermatogenesis Bilateral Zero Zero Zero No Yes 8 Hypospermatogenesis Bilateral 3.7 27.5 2 No No 9 Hypospermatogenesis Unilateral 4.2 32.5 5 No No 10 Maturation arrest Bilateral Zero Zero Zero No Yes 11 Maturation arrest Unilateral Zero Zero Zero No Yes 12 Maturation arrest Unilateral Zero Zero Zero No Yes 13 Maturation arrest Bilateral Zero Zero Zero No Yes 14 Maturation arrest Bilateral 8.9 24 6 Yes No 15 Maturation arrest Unilateral 1.2 37 3 No Yes 16 Maturation arrest Bilateral 2.6 29.5 3 No No 17 Maturation arrest Unilateral Zero Zero Zero No Yes 18 Germ cell aplasia Unilateral Zero Zero Zero No Yes 19 Germ cell aplasia Unilateral Zero Zero Zero No Yes 20 Germ cell aplasia Bilateral Zero Zero Zero No Yes 21 Germ cell aplasia Bilateral Zero Zero Zero No Yes 22 Germ cell aplasia Bilateral Zero Zero Zero No Yes 23 Germ cell aplasia Bilateral Zero Zero Zero No Yes 24 Germ cell aplasia Unilateral 1.8 45.5 4 No Yes 25 Germ cell aplasia Bilateral 7.9 75.7 5 No Yes 26 Germ cell aplasia Unilateral 2.5 35 3 No Yes 27 Germ cell aplasia Bilateral 3.8 32 5 No Yes Pasqualotto. Azoospermic men after varicocelectomy. Fertil Steril 2006. 637

varicocele identified on physical examination may benefit from varicocele repair (13 16, 18 21). Gat et al. (21) observed a significant improvement in sperm concentration, motility, and morphology in 82% of the azoospermic and oligoteratoasthenospermic men after internal spermatic vein embolization. They concluded that if azoospermia is not too long-standing, the treatment of varicocele may significantly improve spermatogenesis or renew sperm production. In addition, adequate treatment may spare in 50% of azoospermic patients the need for TESE as preparation for intracytoplasmic sperm injection (ICSI). On the other hand, Schlegel and Kaufmann (17), evaluating whether a previous history of varicocele repair, although not sufficient to avoid TESE, increases the rate of sperm retrieval, came to conclusion that retrieval rates are 60% per TESE attempt, regardless of whether previous varicocelectomy had been done. The initial benefit of varicocele repair in azoospermic patients treated may be limited, with only 9.6% of men after varicocele repair having enough viable sperm in the ejaculate to avoid a TESE procedure (17). These results for the success rate after varicocelectomy in men with nonobstructive azoospermia is lower than that previously reported (13 16). However, previous reports have considered success to be the presence of sperm on any semen analysis after varicocelectomy (13 16). Schlegel and Kaufmann chose a clinically relevant end point of whether varicocele repair has affected the need for TESE (17). There is another point that might explain why Schlegel and Kaufmann reported less than 10% of men with enough viable sperm in the ejaculate: the presence of subclinical varicocele in their analysis. There are many unresolved clinical questions related to varicoceles. For instance, as to whether subclinical varicoceles should be diagnosed and treated. Therefore, although recent progress in diagnostic methods has revealed a higher incidence of subclinical varicoceles, the clinical significance of this is controversial with regard to male factor infertility (22, 23). Therefore, selecting a population with clinical varicocele might be more suitable to evaluate whether an azoospermic man with varicocele should be operated or not. Another point of discussion is the presence of Y microdeletion or abnormalities in the karyotype, which may be observed in 15% of completely azoospermic men, which may render assisted reproductive technology (ART) necessary (24). According to Cayan et al. (25), azoospermic men undergoing varicocelectomy may achieve improvement in spermatogenesis only when genetic abnormality is not detected. The site of Y chromosome deletion is a more important predictive factor for sperm retrieval than is a coincident varicocele (25, 26). Similarly, for men with Klinefelter syndrome, a history of varicocele repair does not appear to change the outcome of TESE. In the study by Schlegel and Kaufmann 7 patients included in the study had partial deletions of the Y chromosome and 14 patients had Klinefelter syndrome (17). There might be another possible reason for the small percentage of men with enough sperm present in the ejaculate to avoid sperm extraction procedure. In our study, karyotype and Y microdelection were available only in the last 12 patients operated, showing no evidence of abnormalities. The fact that 15 of the 27 patients with left varicocele had right varicocele is of clinical importance. Studying animal models, it was observed that the surgical repair of the secondary right varicocele improved all semen parameters, indicating the harmful consequences of the primary induced left varicocele on the right testis (7). It is important to notice here that in patients with improvement in semen quality, sperm morphology according to the Tygerberg strict criteria varied from 2% to 6%, demonstrating that the varicocele repair may cause an improvement in sperm function. However, despite postoperative improvement in semen parameters in our updated series, ART may still be required to enable the majority of couples to initiate pregnancy (27). The clinical importance of varicocele repair is that a substantial number of completely azoospermic men destined to undergo invasive testicular sperm retrieval in combination with ICSI now have the potential of providing sperm by ejaculation (28, 29). When a choice is possible, using motile spermatozoa from a fresh ejaculate is preferable to using TESE in preparation for ICSI and IVF (27). In addition, couples have to be counseled that they will have to wait for at least 12 months after varicocelectomy before proceeding to any type of ART. Longer follow-up will determine whether these azoospermic patients after the varicocele repair will improve even more their sperm concentration, motility, and morphology. Even without more cases with relapse of azoospermia after an initial appearance of sperm in the semen in our updated series, five of nine patients (55.55%) who had an improvement in the quality of the semen taken 6 months after a varicocele repair returned to their previous azoospermia in a semen analysis performed 12 months after the surgery. We believe that the varicocele repair in those patients who relapsed to azoospermia might have had a temporary effect resulting in the induction of spermatogenesis that, over a small period of time, returned to their original status of azoospermia. On the other hand, there is a possibility that these men have intermittent sperm production and the results are unrelated to the surgery. Nevertheless, these men were not able to sustain spermatogenesis for a long time, suggesting that semen cryopreservation should be performed once the patient has sperm in the semen (13). One may argue that testicular spermatozoa retrieved from azoospermic patients with varicocele might have intrinsic defects, thus reinforcing the idea that these azoospermic patients should undergo a varicocelectomy before initiating any type of ART procedure (25). Again, the importance of these findings is that a significant number of azoospermic 638 Pasqualotto et al. Azoospermic men after varicocelectomy Vol. 85, No. 3, March 2006

men destined to undergo invasive testicular sperm retrieval procedures involving repeated open or needle biopsies in combination with ICSI now have the potential of providing spermatozoa by ejaculation or even of establishing a pregnancy without technical assistance. Although in our updated series the percentage of men with spermatozoa present in the ejaculate after the varicocelectomy had decreased compared to our previous report, we suggest that a varicocele repair must be considered for all men with azoospermia who have a palpable varicocele. A single testis biopsy showing germ cell aplasia may not reflect the overall testis histology, only a focal area. Therefore, azoospermic patients with germ cell aplasia in a single large testis biopsy may have improvement in semen quality after varicocelectomy. 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