Abstract. Introduction. RBMOnline - Vol 19. No Reproductive BioMedicine Online; on web 12 October 2009

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1 RBMOnline - Vol 19. No Reproductive BioMedicine Online; on web 12 October 2009 Article Does age at orchidopexy impact on the results of testicular sperm extraction? Dr Amir Wiser obtained a specialist degree in Obstetrics and Gynecology from Tel Hashomer Hospital at the Sheba Medical Center in Tel Aviv, Israel in He has worked in the IVF Unit at Meir Medical Center, (affiliated with Sackler Faculty of Medicine at Tel Aviv University, Israel), since 2006 as a senior physician. His main research interests are male infertility and polycystic ovary syndrome. Dr Amir Wiser A Wiser 1, G Raviv, R Weissenberg, SE Elizur, J Levron, R Machtinger, I Madgar IVF and Andrology Units, Division of Obstetrics and Gynecology, The Chaim Sheba Medical Centre, Tel Hashomer, Affiliated with the Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel 1 Correspondence: wiserniv@netvision.net.il Abstract The aim of this study was to evaluate the affect of age at the time of orchidopexy on testicular sperm extraction (TESE) results among patients with a history of cryptorchidism and azoospermia. This retrospective study compared TESE results for couples undergoing IVF treatment, among two groups of patients. Group A included patients who underwent orchidopexy at age 10 and younger, and group B included patients who had the procedure above the age of 10. A total of 42 patients were included in the study. Forty patients had bilateral cryptorchidism and two had unilateral. The overall rate of sperm recovery was 59.5%. No differences were found in the sperm retrieval, fertilization, implantation, pregnancy, or live birth rates between the groups. The results suggest that age at orchidopexy, either at 10 years of age or younger or above 10 years of age, was not a predictive factor for successful TESE. Although bilateral cryptorchidism is usually considered a testicular secretory dysfunction, it was found that sperm retrieval attempts yielded spermatozoa in almost 60% of patients with azoospermia and a history of cryptorchidism. Keywords: azoospermia, cryptorchidism, infertility, orchidopexy, testis 778 Introduction The term cryptorchidism is derived from the Greek words kryptos and orchitis, meaning hidden and testis (Murphy et al., 2007). The incidence depends on the gestational age at delivery; approximately 33% of premature male infants are cryptorchid. The incidence decreases to 0.8 1% by 1 year of age (Barthold and González, 2003). The effects of undescended testes and orchidopexy on male fertility are far from clear (Murphy et al., 2007). The long-term results of orchidopexy on a patient s fertility are complicated by other factors such as female fertility, age at orchidopexy, bilateral versus unilateral, preoperative testicular site and testicular size (Murphy et al., 2007). Another variable that can influence a patient s fertility is the prevalence of epididymal or vasal obstructions, which were reported among 17 40% of orchidopexed patients (Nistal et al., 2002; Negri et al., 2003). The recommended age for orchidopexy has become progressively younger, based on histological studies of normal and poorly descended testes (Hedinger, 1982; Cortes et al., 1996; Lee, 2005). Male patients who are diagnosed with cryptorchidism after puberty are more likely to undergo orchidectomy rather than orchidopexy due to evidence that germ cell tumours occur more frequently in undescended testes and postpubertal orchidopexy does not prevent testicular malignancy (Rogers et al., 1998). Cryptorchidism is accountable for 8% of sterility and for 20% of azoospermia (Lee et al., 2001). The only treatment option available for patients with non-obstructive azoospermia (NOA) who would like to father a child with their own spermatozoa is intracytoplasmic sperm injection (ICSI) with testicular sperm extraction (TESE) (Devroey et al., 1995; Tournaye et al., 1995; Schlegel et al., 1997). Patient age at orchidopexy as a positive predictor for successful TESE is still controversial. One study found that Ó 2009 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB23 8DB, UK

2 patient age at orchidopexy was a positive predictor of successful TESE in cryptorchidism patients, and described a higher rate of successful TESE when orchidopexy was performed before age 10 (Raman and Schlegel, 2003). Other studies did not report that age at orchidopexy had any affect on TESE results. Okuyama et al. (1989) found that orchidopexy performed in children 2 5 years of age versus 9 12 years did not result in any differences between the groups in rates of oligospermia and azoospermia. In a more recent study, Hadziselimovic (2006) also found that infertility in cryptorchid patients cannot always be prevented by an early orchidopexy. The aim of this study was to evaluate whether or not age at the time of orchidopexy had any affect on TESE results, implantation or pregnancy rates. Materials and methods Patients All azoospermic patients with a history of cryptorchidism who were referred to the unit from January 1996 to December 2004 were included in this study. Evaluation included semen analysis and physical examination, including evaluation of testicular size by Prader orchidometer. Hormonal analysis included FSH, LH and testosterone concentrations. Karyotype analysis of peripheral leukocytes was performed, including Y-microdeletions. All patients underwent testicular ultrasound and blood tests for malignancy markers that included alpha-fetoprotein (AFP), beta-human chorionic gonadotrophins (b-hcg) and lactate dehydrogenase (LDH). Azoospermia was documented according to the World Health Organization criteria (WHO, 1999) after two semen analyses with an interval of at least 1 month. The semen specimen was centrifuged at high speed (3000 g for 15 min) and subsequent examination of the pellet at high (400) magnification. Patients diagnosed with azoospermia underwent testicular sperm retrieval by TESE. TESE procedure Open, excisional testicular biopsies were taken under general anaesthesia. Scrotal exploration was done via a scrotal or inguinal incision according to the testicular position. An incision of approximately 1 cm was made through the skin and underlying layers. After incision of the tunica albuginea, gentle pressure was applied to the testicular mass and a small specimen of testicular tissue was removed using a pair of curved scissors. Intra-operative wet preparation of the testicular tissue was examined under 200 magnification. If no spermatozoa were identified, further ipsilateral biopsies (usually no more than two or three biopsies to one testis) followed by contralateral biopsies were performed as needed. Some of the testicular tissue was used for histological diagnosis. Testicular tissue samples were collected into sperm wash medium (Cook, Eight-Miles Plains, Queensland, Australia). The germinal epithelium was squeezed out of the seminiferous tubules into the medium with an 18G needle. The cell suspension was transferred into centrifuge tubes and incubated for 2 h in 5% CO 2 at 37 C. After incubation, the suspension was centrifuged for 5 min at 270 g and the pellet was resuspended in about 0.5 ml of sperm wash medium. The suspension was overlaid on two gradient layers of Percoll (Sydney IVF Gradient Kit; Cook) and centrifuged for 20 min at 270 g. The pellet was washed once, resuspended in about 20 ll of medium and dispensed as small aliquots into 10 ll droplets of handling medium under oil for sperm collection and ICSI. When at least a single spermatozoa was identified in each microscopic field (200) according to Hauser s scale 2 (Hauser et al., 1998), the excess spermatozoa were cryopreserved. Diagnostic testicular biopsy was prepared with haematoxylineosin stain and histopathology classified according to the following parameters: (i) normal spermatogenesis; (ii) hypospermatogenesis; (iii) complete maturation arrest (MA); and (iv) pure sertoli cell only (SCO) (Tournaye et al., 1997). A quantitative evaluation of mature spermatids (mean number of completely elongated spermatids in at least 20 sections of tubules) was also performed. Hypospermatogenesis was arbitrarily classified as the observation of fewer than six mature spermatids per seminiferous tubule cross-section (Silber et al., 1997). IVF protocol All female patients were treated simultaneously with the long IVF protocol, starting with down-regulation on day 21 of menstruation with the gonadotrophin-releasing hormone (GnRH) agonist triptorelin acetate (i.m. decapeptyl CR 3.75 mg or daily s.c. decapeptyl 0.1 mg; Ferring GmbH, Germany). When down-regulation was achieved, stimulation was started with human menopausal gonadotrophins (Menogon; Ferring SA, Sainet-Prex, Switzerland) or rfsh (Gonal; F Merck Serono SA, Aubunne, Switzerland or Purigon; NV Organon Oss, The Netherlands). Oocyte retrieval was scheduled 36 h after injection of 10,000 IU of purified urinary HCG (Pregnyl; Organon Oss) or 250 lg of recombinant HCG (Ovitrel; Merck Serono SA, Bari, Italy). Just prior to oocyte retrieval, TESE was performed and when positive sperm retrieval was achieved, oocyte retrieval was accomplished. The oocytes were cultured individually in drops under mineral oil (Cook, Australia). In all cycles, the fertilization was by ICSI. Only metaphase II oocytes were injected with preferably motile spermatozoa into the ooplasm. Fertilization was assessed 20 h after insemination for the appearance of pronuclei. Up to two best-quality embryos were transferred on day 2 or 3. Clinical pregnancy was confirmed by a fetal heartbeat, determined by transvaginal ultrasound approximately 32 days after embryo transfer. Statistical analysis For data analysis, patients were divided into two groups according to age at the time of orchidopexy: group A consisted of patients who underwent orchidopexy at age 10 or under and group B those who had the procedure at above age 10. Categorical variables were compared by chi-squared test and baseline characteristics were compared between the groups using one-way analysis of variance (ANOVA). The relationship between variables was calculated using the Pearson correlation analysis. The statistical significance level was set at P

3 Table 1. Characteristics of patients according to age at orchidopexy. Patient characteristic Age at orchidopexy Group A: 0 10 years (n = 21) Group B: >10 years (n = 21) Age at TESE (years) 33.7 ± ± 6.8 FSH (IU/l) 20.1 ± ± 18.4 LH (IU/l) 10.2 ± ± 1.3 Testosterone (nmol/ml) 15.5 ± ± 5.1 Right testis size (ml) 14.2 ± ± 7.5 Left testis size (ml) 14.4 ± ± 7.0 Histology Hypospermatogenesis 0 2 Maturation arrest (MA) 4 0 Sertoli cell only (SCO) 5 13 Combined (SCO + MA) 6 3 No data 6 3 Values are mean ± SD or n. There were no statistically significant differences between the two groups. TESE = testicular sperm extraction. Results Forty-two patients were included in the study. Forty had bilateral cryptorchidism and two had unilateral cryptorchidism. The patients ages at orchidopexy ranged between 1 and 21 years old (mean 10.0 ± 7.1 years). There were 21 patients in group A (those who were operated on at age 10 or younger) and 21 patients in group B (operated on above the age of 10). All patients had normal male karyotype, 46XY without Y-microdeletions. Testicular biopsy results were: two patients with hypospermatogenesis, four with maturation arrest (MA), 18 with Sertoli cell only (SCO), and nine with combined SCO and MA. In nine patients, the histology was missing. There were no significant differences between the two groups regarding age at testicular biopsy, 33.7 ± 5.2 and 33.1 ± 6.8 years respectively. No significant differences were found in FSH, LH or testosterone concentrations, or testicular volume between the groups (Table 1). Only three patients in group A and two patients in group B had normal FSH concentrations ( miu/ml), the remaining patients had FSH concentrations above the normal range. The number of patients with low orchidometry (larger testis < 15 ml) was also similar between the two groups. The total rate of sperm recovery was 59.5%, with no significant differences between group A (61.9%) and group B (57.1%). In 20 patients, the spermatozoa remaining after ICSI were of appropriate quality and were cryopreserved. The number of oocytes retrieved from the partners of groups A and B was not significantly different at 11.8 ± 5.1 and 15.5 ± 6.0 respectively. The fertilization rates were also not significantly different between the groups at 53.4% and 48%, respectively (Table 2). The implantation rate in group A was 22.2%, not significantly different from the rate for group B of 26.1%. The clinical pregnancy rates (from patients with spermatozoa during TESE) were 30.8% (4/13 in group A) and 41.2% (5/12 in group B). Subsequent live birth rates were 75% (3/4) in group A and 80% (4/5) in group B (Table 2). Table 3 shows the differences between patients in whom spermatozoa were found compared with those with no spermatozoa. In group A, the patients with successful sperm Table 2. IVF cycle characteristics according to age at orchidopexy. Cycle characteristic Age at orchidopexy Group A: 0 10 years (n = 21) Group B: >10 years (n = 21) Spermatozoa recovery rate (%) 13 (61.9) 12 (57.1) Mean no. oocytes retrieved 11.8 ± ± 6.0 Fertilization rate% Female age (years) 29.0 ± ± 5.9 Implantation rate (%) Clinical pregnancy rate (%) 4/13 (30.8) 5/12 (41.2) Live births/clinical pregnancy (%) 3/4 (75.0) 4/5 (80.0) 780 There were no statistically significant differences between the two groups.

4 Table 3. Characteristics of patients according age at orchidopexy and recovery of spermatozoa. Patient characteristic Recovery of spermatozoa P-value Group A: 0 10 years (n = 21) Spermatozoa found (n = 12) Spermatozoa not found (n =9) FSH (IU/l) 16.6 ± ± LH (IU/l) 9.5 ± ± Testosterone (nmol/ml) 17.4 ± ± 4.0 NS Right testis size (ml) 17.0 ± ± Left testis size (ml) 14.8 ± ± 2.9 NS Group B > 10 years (n = 21) Spermatozoa found (n = 12) Spermatozoa not found (n =9) FSH (IU/l) 28.1 ± ± 10.3 NS LH (IU/l) 11.8 ± ± 8.0 NS Testosterone (nmol/ml) 14.3 ± ± 4.8 NS Right testis size (ml) 15.1 ± ± 4.8 NS Left testis size (ml) 13.4 ± ± 8.2 NS Groups A and B combined (n =42) Spermatozoa found (n = 24) Spermatozoa not found (n = 18) FSH (IU/l) 22.2 ± ± 9.1 NS LH (IU/l) 10.6 ± ± 6.3 NS Testosterone (nmol/ml) 15.8 ± ± 4.5 NS Right testis size (ml) 16.0 ± 0.o 11.0 ± Left testis size (ml) 14.1 ± ± 3.4 NS Values are mean ± SD. NS = not statistically significant. retrieval had significantly lower FSH concentrations, 16.6 ± 11.6 IU/ml versus 26.3 ± 7.7 IU/ml (P < 0.05) and larger right testis volume 17.0 versus 10.3 ml respectively (P = 0.05). However, these findings were not observed among the patients in group B. Discussion The development of ICSI offers a therapeutic strategy for patients with NOA, including those associated with cryptorchidism (Shin et al., 1997; Giwercman et al., 2000). However, this procedure is not successful in finding spermatozoa that can be used for ICSI for all patients (Negri et al., 2003; Raman and Schlegel, 2003). Looking for predictive factors for successful TESE among azoospermic patients with a history of cryptorchidism, it was speculated whether the age at time of orchidopexy had any impact on the possibility of sperm recovery. Several studies have focused on patient age at orchidopexy. Okuyama et al. (1989) compared bilateral orchidopexy at 2 5 years of age versus 9 12 years of age and did not find differences between the groups in the rates of oligozoospermia and azoospermia. Raman and Schlegel (2003) described a significantly higher TESE success rate of 94%, in patients who underwent orchidopexy between birth and 10 years of age, compared with patients who underwent orchidopexy between 11 and 20 years of age, and above age 20, at only 43% and 44%, respectively. The study has added data to the literature about age at orchidopexy and potential fertility. By 6 8 months of age, undescended testes show a delay in germ cell development and maturation, specifically in the transformation of gonocyte into adult dark (Ad) spermatogenia and a delay in the appearance of primary spermatocytes among cryptorchidic testes (Rogers et al., 1998). A decrease in germ cells in those who underwent orchidopexy before or after 6 months of age was also observed (Hadziselimovic and Herzog, 2001). According to the data, age at orchidopexy had no impact on the probability of finding spermatozoa. However in this study, similar to previous studies that focused on age at orchidopexy, the patients were surgically treated for undescended testes after the potential damage had probably occurred. The earliest orchidopexy in this study was performed at 1 year of age, and only four patients had been operated on before age 4. Recent evidence of damage to the testes in the first months of life has influenced the recommended age for orchidopexy. It is generally assumed that spontaneous descent is rare beyond 6 months of age (Wenzler et al., 2004). The current recommendation is for orchidopexy between 6 and 9 months (Lee et al., 2005). The mechanism of damage during the first months of life could explain why there was no difference in spermatozoa findings between the present study groups and in other studies. High FSH concentrations measured in the two groups (Table 1) probably also support the theory of early damage to the testes. The effect of early surgery (before 1 year of age) on fertility outcomes remains to be seen in years time, when the present generation of children with early orchidopexy reaches adulthood. 781

5 782 An attempt was made to find other predictors for sperm recovery (Table 3). The study groups were divided according to spermatozoa findings. In group A, FSH was lower among patients with successful TESE, 16.6 compared with 26.3 IU/l in patients without sperm recovery (P < 0.05). This finding was not observed among patients in group B. High FSH concentrations in patients with and without spermatozoa findings have also been described by other authors (Raman and Schlegel, 2003). Other studies found FSH to be a positive predictor for successful sperm retrieval (Andersson et al., 2004; Kojima et al., 2006). Testicular volume was also described as a positive predictive factor by some authors (Raman and Schlegel, 2003; Kojima et al., 2006) and as an unreliable factor by others (Su et al., 1999). In this group of patients, there were no differences in testicular volume according to age at orchidopexy (Table 1). However, the mean testicular volume was below average for all patients. When the patients were grouped according to positive or negative TESE results (Table 3), there was also no difference in testicular volume; however, in group A, patients with successful TESE had a larger right testis volume (P = 0.05). Kollin et al. (2007) recently found that early orchidopexy, at approximately 9 months of age, resulted in an increased testicular volume compared with patients who had orchidopexy at age 3, and hoped that early orchidopexy may improve future spermatogenesis. FSH concentrations and testicular volumes seem to be positive predictors for sperm recovery. The variability of the findings between groups A and B could be due to the relatively small number in each group. The early orchidopexy approach appears desirable, but for patients who were not operated on during childhood and were diagnosed with cryptorchidism in adolescence, testicular preservation for future fertility seems preferable to orchidectomy. Two patients in the present group who were diagnosed with undescended testes during the infertility evaluation underwent successful TESE. This approach is in contrast to Rogers et al. (1998), who found that cryptorchidic testes in postpubertal males do not contribute to fertility and have significant malignant potential. A thorough work-up to rule out malignancy is needed. The authors current policy is to perform a physical examination, testicular ultrasound and blood testing for malignancy markers (AFP, b-hcg and LDH). No case of malignancy was diagnosed among the 42 patients in this study. In conclusion, the present results suggest that age at orchidopexy (between 1 and 10 years versus above 10 years of age) is not a predictive factor for successful TESE. These results seem to support the current argument for orchidopexy before 1 year of age. Although bilateral cryptorchidism is usually considered a testicular secretive dysfunction, it was found that sperm retrieval attempts yielded spermatozoa in almost 60% of patients with azoospermia and a history of cryptorchidism. References Andersson AM, Petersen JH, Jørgensen N et al Serum inhibin B and follicle-stimulating hormone levels as tools in the evaluation of infertile men: significance of adequate reference values from proven fertile men. Journal of Clinical Endocrinology and Metabolism 89, Barthold JS, González R 2003 The epidemiology of congenital cryptorchidism, testicular ascent and orchiopexy. Journal of Urology 170, Cortes D, Thorup JM, Lindenberg S 1996 Fertility potential after unilateral orchiopexy: simultaneous testicular biopsy and orchiopexy in a cohort of 87 patients. Journal of Urology 155, Devroey P, Liu J, Nagy Z et al Pregnancies after testicular sperm extraction and intracytoplasmic sperm injection in nonobstructive azoospermia. 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6 Su LM, Palermo GD, Goldstein M et al Testicular sperm extraction with intracytoplasmic sperm injection for nonobstructive azoospermia: testicular histology can predict success of sperm retrieval. Journal of Urology 161, Tournaye H, Verheyen G, Nagy P et al Are there any predictive factors for successful testicular sperm recovery in azoospermic patients? Human Reproduction 12, Tournaye H, Camus M, Goossens A et al Recent concepts in the management of infertility because of non-obstructive azoospermia. Human Reproduction 10(Suppl.), Wenzler DL, Bloom DA, Park JM 2004 What is the rate of spontaneous testicular descent in infants with cryptorchidism? Journal of Urology 171, World Health Organization 1999 WHO Laboratory Manual for the Examination of Human Semen and Sperm (Cervical Mucus Interaction, 4th ed. Cambridge University Press, Cambridge, UK. Declaration: The authors report no financial or commercial conflicts of interest. Received 28 January 2009; refereed 20 March 2009; accepted 7 September