Outcome of testicular sperm recovery and ICSI in patients with non-obstructive azoospermia with a history of orchidopexy

V. Vernaeve1,2, A. Krikilion1, G. Verheyen1, A. Van Steirteghem1, P. Devroey1 and H. Tournaye1

Centre For Reproductive Medicine, University Hospital, Dutch-speaking Brussels Free University (Vrije Universiteit Brussel), Laarbeeklaan 101, B-1090, Belgium

2 To whom correspondence should be addressed. Email: valerievernaeve{at}yahoo.fr


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Little is known about sperm recovery and ICSI using testicular sperm from men with non-obstructive azoospermia who had a previous orchidopexy. We therefore studied the sperm recovery in this subgroup and evaluated clinical parameters predicting successful sperm retrieval and the outcome of ICSI. METHODS: A total of 79 non-obstructive azoospermic men with a history of orchidopexy underwent a sperm recovery procedure. The predictive value of clinical parameters such as age at sperm retrieval, age at orchidopexy, testicular volume, FSH, FSH/LH ratio, testosterone and androgen sensitivity index (LH x testosterone) for successful testicular sperm retrieval was evaluated using receiver operating characteristics (ROC) curve analysis. A comparison between 64 ICSI cycles performed in these couples and 92 cycles performed in couples in which the men had an unexplained non-obstructive azoospermia was carried out. RESULTS: Testicular spermatozoa were recovered in 41 patients (52%). The mean age at orchidopexy of the patients with a positive sperm recovery was 10.6 years [95% confidence interval (CI) 7.3–13.8] versus 15.5 years (95% CI 11.3–19.8) for those where no spermatozoa were found. The mean testicular volume of the largest testis of patients with spermatozoa found was 10 ml (95% CI 8.3–11.9) versus 8.5 ml (95% CI 5.8–11.1) in patients with no spermatozoa found. The mean FSH and testosterone value for patients with successful and unsuccessful sperm recovery, respectively, was 24.1 IU/l (95% CI 17.9–30.3) and 4.4 ng/ml (95% CI 3.7–5.1) versus 28.8 IU/l (95% CI 19.4–38.2) and 3.4 ng/ml (95% CI 2.2–4.5). All clinical and biological parameters examined failed to predict the outcome of the testicular sperm extraction. No differences were observed between the orchidopexy and unexplained group for the number of oocytes retrieved, fertilization rate, embryo quality, pregnancy rate and implantation rate. CONCLUSIONS: As in the population of men with non-obstructive azoospermia, the sperm recovery rate for patients with a history of orchidopexy is ~50% and there are currently no clinical parameters predicting successful sperm retrieval in this subpopulation of patients. The outcome of the ICSI cycles is comparable with that in the population of men with non-obstructive azoospermia.

Key words: cryptorchidism/ICSI/non-obstructive azoospermia/orchidopexy/TESE


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Cryptorchidism is a common cause of male infertility, with a prevalence of 1% in adults, 3–8% in infertile men and 20% in men with azoospermia (Lee and Coughlin, 2001Go). The aetiology of this disorder is not completely understood. A functional hypothalamo-pituitary–gonadal axis is a prerequisite for a normal descent of the testes. As early as the first months after birth, histological anomalies are observed in cryptorchid testes (Hedinger, 1982Go), which may lead to a primary testicular failure. If a man has a non-obstructive azoospermia (NOA) and wants to father his own genetic child, the only treatment option available for the couple is ICSI with testicular sperm (TESE) (Devroey et al., 1995Go; Tournaye et al., 1995Go; Silber et al., 1996Go; Schlegel et al., 1997Go). However, testicular biopsy in NOA patients is not always successful in terms of finding testicular spermatozoa. An unsuccessful sperm recovery procedure has important emotional and financial implications as well as physiological complications such as devascularization and fibrosis of the testis (Friedler et al., 1997Go; Schlegel and Su, 1997Go; Ron-El et al., 1998Go). Objective counselling of the azoospermic patient is therefore of utmost importance. Several small-scale reports have described successful pregnancies after TESE with ICSI for men with NOA associated with cryptorchidism (Shin et al., 1997Go; Giwercman et al., 2000Go; Lin et al., 2001Go; Negri et al., 2003Go; Raman and Schlegel, 2003Go).

The present study presents a large patient population with a history of orchidopexy with the exclusion of any other pathology that could explain testicular failure and histologically proven NOA.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Patients
We reviewed the files of all NOA patients who had testicular sperm recovery for ICSI in the period from January 1995 to December 2002. All patients had at least two semen analyses including a centrifugation step at high speed. They all had a clinical work-up including physical examination, hormonal assessment (FSH, LH and testosterone), assessment of biochemical markers (fructose and {alpha}-glucosidase), karyotype analysis and assessment for Yq microdeletion. Scrotal ultrasound was performed whenever indicated. As in the study by Matsumiya et al. (1994)Go, the group of patients with NOA included patients with incomplete spermatogenesis. The histology of these patients showed maturation arrest with or without focal spermatogenesis, germ cell aplasia (Sertoli cell-only syndrome) with or without focal spermatogenesis, and tubular sclerosis and atrophy. In 213 patients, the medical history was uneventful and the work-up was normal. They were defined as having an unexplained NOA. Seventy-nine NOA patients had a history of unilateral or bilateral orchidopexy. Patients with antecedents of cryptorchidism who had no orchidopexy were not included in this series. Of the 79 patients with a history of orchidopexy, 49 (62%) had a history of bilateral orchidopexy, 16 (20.3%) of unilateral orchidopexy, and in 14 cases (17.7%) this information was not clear and not verifiable.

Serum LH and FSH were measured using the automated Elecsys 2010 immunoanalyser (Roche Diagnostics, Mannheim, Germany). Intra- and inter-assay coefficients of variation were <3 and <4% for LH, and <3 and <6% for FSH, respectively.

Testosterone was measured using the Spectria Testosterone coated tube radioimmunoassay (Orion Diagnostica, Espoo, Finland).

The androgen sensitivity index (LH x testosterone) was calculated according to Kamischke et al. (2003)Go.

Testicular sperm recovery
Open excisional testicular biopsies were taken under general anaesthesia as described previously by Tournaye et al. (1997)Go, either on the day of ovum retrieval or during a preliminary surgery with a view to cryopreservation. An incision of ~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 the protruding testicular mass was removed using a pair of curved scissors. The weight of each tissue sample was ~150 mg. The testicular tissue was placed in a Petri dish containing HEPES-buffered Earle's medium supplemented with 0.5% human serum albumin (Red Cross, Brussels, Belgium) and taken to the adjacent laboratory. In the laboratory, the wet preparation of testicular tissue was shredded roughly using two microscope glass slides in a Petri dish (3002; Becton-Dickinson, Aalst, Belgium) on the warmed stage of a stereomicroscope at x40 magnification (Verheyen et al., 1995Go). During this procedure, the seminiferous tubules were unravelled and broken. The tissue was then minced further with two fine forceps (Lawton, Tuttlingan, Switzerland) in a Petri dish (‘mincing’), until tissue pieces of ~1 mm3 or free tubuli pieces of a few millimetres in length were obtained. Under an inverted microscope (x400 magnification), the shredded tissue was then checked for the presence of spermatozoa. When spermatozoa or late elongated spermatids (stage Sd2) according to Clermont (1963)Go were found on wet preparations of the biopsies, surgery was discontinued. Otherwise, at least four biopsies were taken randomly on each side. Since 1998, enzymatic digestion of the testicular tissue with collagenase type IV has been performed whenever sperm were not found after mechanical shredding (Crabbé et al., 1998Go). During surgery, a randomly taken, single, small biopsy was sent for histopathological examination.

Ovarian stimulation and ICSI procedure
The outcome of ICSI cycles using fresh or frozen–thawed sperm, performed in patients with a history of orchidopexy, was compared with the ICSI cycles using fresh sperm, performed in patients with unexplained NOA.

All female partners were superovulated using a GnRH analogue suppression protocol and HMG. Oocyte–cumulus complexes were recovered 36 h after the administration of 10 000 IU of HCG. After removal of cumulus cells, the nuclear maturation of the oocytes was assessed under an inverted microscope. Only metaphase II oocytes were injected with preferably a motile spermatozoon into the ooplasm. These procedures have been described previously (Van Steirteghem et al., 1993Go; Joris et al., 1998Go).

Assessment of fertilization, embryo cleavage and establishment of pregnancy
Further culture of injected oocytes was performed in 25 µl microdrops of culture medium under lightweight paraffin oil. Fertilization was confirmed after 16–18 h by the observation of two distinct pronuclei under an inverted microscope. Developing embryos were classified according to their morphological appearance. Cleaving embryos with <50% of their volume filled with anucleate fragments were considered eligible for transfer. Cleaving embryos were transferred into the uterine cavity 2 or 3 days after the ICSI procedure.

A rise in serum HCG on two consecutive occasions from 11 days after embryo transfer indicated pregnancy. Each pregnancy with at least one fetus with positive heartbeat revealed by ultrasonography ~5 weeks after transfer was considered as a clinical pregnancy. The implantation rate was defined as the ratio of fetuses with heartbeat to the number of embryos transferred. Pregnancies with unknown outcome were considered non-clinical.

Statistical analysis
The predictive power of clinical parameters such as age, testicular volume, FSH, FSH/LH ratio, testosterone and androgen sensitivity index for the two groups, i.e. successful and unsuccessful sperm recovery, were evaluated by receiver operating characteristics (ROC) analysis (Zweig and Campbell, 1993Go) using Medcalc (Medcalc Software, Ghent, Belgium). ROC graphs are plots of all the sensitivity (y-axis) and specificity (x-axis) pairs which are possible for all levels of a particular parameter. Calculation of the area under the curve provides a quantitative measure of accuracy, i.e. the ability of this parameter to discriminate between two conditions. A test with perfect discrimination has an ROC plot that passes through the upper left-hand corner, where the true positive fraction is 1.0 or 100% (maximal sensitivity) and the false-positive fraction is 0 (maximal specificity). The theoretical plot for a test with no discrimination (identical distributions of results for the two groups) is a 45° diagonal line from the lower left-hand corner (0% true-positive rate and 0% false-positive rate) to the upper right-hand corner (100% true-positive rate and 100% false-positive rate) with an area under the curve of 0.5 (Zweig and Campbell, 1993Go).

Comparisons for qualitative variables were performed using Fisher's exact test. A Mann–Whitney test was used when data were not normally distributed. A P-value of <0.05 was considered to be statistically significant.

This observational study was approved by our institutional review board.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Sperm recovery
Among the 79 patients with orchidopexy who underwent their first TESE, 54 (68.4%) showed complete or incomplete germ cell aplasia (Sertoli cell-only) at testicular histopathology, 20 (25.3%) showed complete or incomplete maturation arrest and five (6.3%) showed tubular sclerosis and atrophy.

Testicular spermatozoa were recovered in 41 of these 79 patients (51.9%) [95% confidence interval (CI) 40.9–62.9]. Table I shows the sperm recovery in the different histological subcategories.


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Table I. Testicular sperm recovery in patients with a history of orchidopexy in the different histological subcategories

 
Testicular spermatozoa were recovered in 21 of the 49 patients (43%) with bilateral and in nine out of the 16 (56.2%) with unilateral cryptorchidism (P=0.4). The mean age of all patients at testicular biopsy was 34.0 years (95% CI 32.1–35.8). The mean age of those with spermatozoa found was 32.0 years (95% CI 29.7–34.4) versus 35.1 years (95% CI 32.3–37.8) for those with no spermatozoa found. The mean age at orchidopexy of the patients with spermatozoa found was 10.6 years (95% CI 7.3–13.8) versus 15.5 years (95% CI 11.3–19.8) for those with no spermatozoa found. The mean testicular volume of the largest testis of patients with spermatozoa found was 10 ml (95% CI 8.3–11.9) versus 8.5 ml (95% CI 5.9–11.1) in patients with no spermatozoa found. The mean FSH value for patients with spermatozoa found was 24.1 IU/l (95% CI 17.9–30.3) versus 28.8 IU/l (95% CI 19.4–38.2) for patients with no spermatozoa found. The mean testosterone value for patients with spermatozoa found was 4.4 ng/ml (95% CI 3.7–5.1) versus 3.4 ng/ml (95% CI 2.2–4.5) for patients with no spermatozoa found. None of these parameters was able to predict the outcome of a testicular sperm extraction procedure (Figure 1 and Table II). In 76 patients, detailed information was available as to the presence or absence of spermatids or spermatozoa on histopathology. In all patients where spermatozoa were found at histology, spermatozoa were also found on wet preparation (n=13). There were no cases of patients with spermatozoa found on histopathology but not on wet preparation. Twenty-five patients had no spermatozoa found on histopathology but had a successful TESE. For 38 patients, there were no spermatozoa on histology and none after TESE. Histology had a sensitivity of 34.2%, a specificity of 100%, a positive predictive value of 100% and a negative predictive value of 60.3%.



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Figure 1. Receiver operating characteristics (ROC) curves of clinical and biological parameters for discriminating successful and failed testicular sperm extraction in non-obstructive azoospermic patients with a history of orchidopexy. The best discriminating values are indicated.

 

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Table II. Clinical and biological parameters for discriminating successful and failed testicular sperm extraction in non-obstructive azoospermic patients with a history of orchidopexy

 
Comparison between patients with a history of orchidopexy and patients with unexplained non-obstructive azoospermia
In the same study period, 213 men with unexplained NOA had their first TESE procedure. In 71 of these men (33.3%), testicular sperm was recovered successfully (95% CI, 27.0–39.7).

Sixty-seven of these men underwent 92 ICSI cycles using fresh sperm. Their mean age at testicular biopsy was 35.2 years (95% CI, 34.0–36.4).

Of the 41 patients with a history of orchidopexy and with a positive sperm recovery, 37 underwent 64 ICSI cycles with fresh or frozen–thawed sperm. In 46 ICSI cycles, fresh sperm was used, and in 18 frozen–thawed testicular sperm was used. The outcome of these cycles was compared with 92 ICSI cycles performed on 67 patients using fresh sperm and performed during the same period on patients with an unexplained NOA. The mean age of the female partners at oocyte retrieval was 31.1 years (95% CI, 29.4–32.7) versus 32.6 years (95% CI, 31.1–34.2) in the orchidopexy and idiopathic group, respectively. In the 64 cycles performed on patients with orchidopexy and 92 cycles on patients with unexplained infertility, the average number of oocytes retrieved per cycle was 12.3 versus 12.7, respectively (NS). The mean of the percentages of oocytes showing two pronuclei was 50.5 versus 52.2%, respectively (NS). On average, 72.4 versus 67.6% of the embryos were of good morphological quality in the respective groups (NS) (Table III). The pregnancy rates per embryo transfer were 34 and 22.6% (NS) for the orchidopexy and unexplained group, respectively. The clinical pregnancy rates per embryo transfer were 20.8 versus 11.9% (NS) in the respective groups. The implantation rate per embryo was 9.8 in the orchidopexy and 6.0% in the unexplained group (NS) (Table IV). Eleven deliveries resulted in the birth of 15 live born children in the orchidopexy group. In the unexplained group, there were 10 deliveries of 11 children. Two of these children were stillborn. These two children were part of two different multiple pregnancies. One twin pregnancy ended prematurely at 24 weeks and resulted in a stillborn child with a polymalformative syndrome (fused eyes, imperforated anus and ambiguous sex). The other part of this twin pregnancy died 2 weeks after birth because of the pre-term birth complications. The other stillborn child was part of a triplet pregnancy that was reduced to a twin pregnancy in the first trimester of the pregnancy. One of the two remaining fetuses was stillborn; the other was a live born child. In the 15 live born children in the orchidopexy group, one major (lipomeningocoele) and one minor (open ductus arteriosus) malformation were observed. No malformations were observed in the live born children in the unexplained group.


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Table III. Fertilization, cleavage rate and pregnancies in patients with NOA with a history of orchidopexy versus unexplained NOA

 

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Table IV. Pregnancies in and children born to patients with NOA with a history of orchidopexy versus unexplained NOA

 
In six cycles (five couples), frozen–thawed embryos were transferred in couples in which the husband had a history of orchidopexy, which resulted in one live born child.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Recovery of testicular spermatozoa (TESE) for ICSI is a useful therapeutic strategy for patients with NOA associated with cryptorchidism (Shin et al., 1997Go; Giwercman et al., 2000Go; Lin et al., 2001Go). However, this procedure is not successful in all patients in terms of finding spermatozoa that can be used for ICSI (Negri et al., 2003Go; Raman and Schlegel, 2003Go).

We examined a consecutive series of 79 patients with histologically proven NOA and with orchidopexy in their history. Testicular spermatozoa were recovered successfully in 41 of these 79 patients (51.9%) (95% CI 40.9–62.9). This recovery rate is in accordance with previous reports from our centre (Tournaye et al., 1995Go; Vernaeve et al., 2002Go). However, this percentage is significantly higher than that observed in unexplained NOA. In this subgroup, testicular spermatozoa were recovered successfully in 71 out of the 213 patients (33.3%) (95% CI 27.0–39.7). This higher recovery rate in patients with a history of orchidopexy may be explained by the fact that some of these patients may have been misdiagnosed in their youth as having undescended testis when in fact they may have had a retractile testis (Hack et al., 2003Go). Although generally regarded as not requiring any therapy (Schoorl, 1982Go), some authors describe retractile testes as a risk factor for subfertility and suggest orchidopexy (Caucci et al., 1997Go). We tried to correct for this well-known diagnostic pitfall by excluding patients with a history of orchidopexy and a testicular histopathology showing normal spermatogenesis. Negri et al. (2003)Go confirmed this by finding a high prevalence of histological patterns compatible with an obstruction in patients with antecedents of bilateral orchidopexy. The testicular sperm extraction rate in our patients with a history of cryptorchidism is lower than the 74% reported by Raman and Schlegel (2003)Go. Differences in the population studied may account for this. The Raman series included patients with other co-existent pathologies, i.e. varicocoelectomy, Klinefelter's syndrome, Yq microdeletion and chemotherapy. The absence of testicular pathology in some patients may also explain their higher recovery rate as orchidopexy performed in childhood is known to be associated with seminal duct anomalies (from rete testis to vas deferens) or with iatrogenic obstruction by transection or excessive tension in the spermatic cord (Adamsen and Borjesson, 1988Go; Redman, 2000Go). However, the most important factor that may explain the high recovery rate in their series may be the preliminary diagnostic testicular biopsies performed on almost all their patients before embarking on TESE (Raman and Schlegel, 2003Go). Our results do in fact show that a high recovery rate can be expected when histology shows spermatids or spermatozoa. Other parameters, however, fail to predict the recovery, i.e. age at testicular biopsy, age at orchidopexy, testicular volume, FSH, FSH/LH ratio and androgen sensitivity index. However, it may not be excluded that with a larger sample size some predictors may acquire discriminative power.

We did not find any difference in the outcome of the ICSI cycles and the pregnancy rates between patients with orchidopexy and patients with unexplained NOA. Although the ICSI results in the present subgroups are similar to those in our NOA population reported earlier (Vernaeve et al., 2003Go), the pregnancy and implantation rates are lower than in the series reported by Raman and Schlegel (2003)Go. This again may be explained by the inclusion of patients with normal spermatogenesis or hypospermatogenesis at histology in the latter study.

In both groups, a high rate of early pregnancy loss was observed. This is in agreement with the findings in our larger series of NOA patients (Vernaeve et al., 2003Go).

In the present series, none of the children born had a cryptorchidism. In the future, however, it would be worthwhile evaluating, in a larger series, the prevalence of cryptorchidism in boys born from fathers with an undescended testis.

In conclusion, the sperm recovery rate for a man with a record of orchidopexy is comparable with the recovery rate for the population of NOA men. Unfortunately, no clinical predictive factors for successful testicular sperm extraction are, so far, available either for the overall NOA population or for the subpopulation of men with a history of orchidopexy.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We would like to thank the clinical, paramedical and laboratory staff of the Centre for Reproductive Medicine. Furthermore, we are very grateful to Mr Michael Whitburn of the Language Education Centre of our University for proof-reading our paper. The research work was supported by grants from the Fund for Scientific Research-Flanders (FWO-Vlaanderen).


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on February 10, 2004; accepted on June 4, 2004.