Cumulative delivery rates after ICSI treatment cycles with freshly retrieved testicular sperm: a 7-year follow-up study

Kaan Osmanagaoglu1, Valerie Vernaeve, Efstratios Kolibianakis, Herman Tournaye, Michel Camus, Andre Van Steirteghem and Paul Devroey

Center for Reproductive Medicine, Dutch-speaking Brussels Free University, Brussels, Belgium

1 To whom correspondence should be addressed. e-mail: osmanagaoglu{at}hotmail.com


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: The purpose of this study was to assess cumulative delivery rates in patients with non-obstructive or obstructive azoospermia following treatment by testicular sperm extraction (TESE)–ICSI. METHODS: A cohort follow-up study was conducted. Between January 1994 and December 2000, 364 couples with obstructive azoospermia underwent a total of 609 fresh TESE–ICSI treatment cycles. In addition, 303 fresh TESE–ICSI treatment cycles were performed in 235 couples for non-obstructive azoospermia. This study included only patients in whom sperm was recovered. In the non-obstructive group, only patients with maturation arrest, atrophic sclerosis and germ cell aplasia were included. The main outcome measure was a delivery beyond 25 weeks gestation. RESULTS: In patients with obstructive azoospermia, the crude delivery rate after three cycles was 35% while the expected cumulative delivery rate was 48% [95% confidence interval (CI), 41–55]. On the other hand, in patients with non-obstructive azoospermia, the crude cumulative delivery rate after three treatment cycles was 17% while the expected delivery rate was 31% (95% CI, 15–46). A high dropout rate in couples with both non-obstructive and obstructive azoospermia was observed (75 and 50% respectively, after the first cycle). CONCLUSION: This study shows that there is a value in performing several TESE–ICSI attempts in patients with obstructive and non-obstructive azoospermia. The estimates of the non-obstructive group beginning from the third cycle are less reliable due to fewer patients. However, overall, the obstructive group performed better than the non-obstructive group.

Key words: cumulative pregnancy rates/life-table/non-obstructive azoospermia/obstructive azoospermia/TESE-ICSI


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The introduction of IVF embryo transfer in 1978 (Steptoe and Edwards, 1978Go) and ICSI in 1991 (Palermo et al., 1992Go; Van Steirteghem et al., 1993Go) helped many infertile couples to be treated successfully. However, many patients who did not have sperm in their ejaculate could not be helped by the ICSI technique. Testicular sperm extraction (TESE) opened up new perspectives for such couples (Schoysman et al., 1992Go; Craft et al., 1993Go; Devroey et al., 1994Go, 1995).

Although significant progress has been recorded in reproductive medicine, multiple assisted reproductive technique (ART) cycles are still necessary (Kolibianakis et al., 2002Go) in order to achieve a live birth. For this reason, the need to inform the patients about the chances of delivery following consecutive cycles is evident.

Cumulative delivery rates after ICSI using freshly ejaculated sperm have already been reported in patients younger (Osmanagaoglu et al., 1999Go) or older than 37 years (Osmanagaoglu et al., 2002Go). Success rates for the TESE technique have been reported per ICSI cycle, but so far no cumulative success rates are available for cumulative delivery rates after ICSI with surgically retrieved sperm, making appropriate counselling rather difficult. The few data available in this respect do not discriminate between obstructive and non-obstructive azoospermia.

This study aims to assess the cumulative delivery rates in patients with non-obstructive and obstructive azoospermia following treatment by ICSI using freshly retrieved testicular sperm. We aimed to analyse the efficiency of consecutive interventions for testicular sperm retrieval in an assisted reproduction programme.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients
A total of 912 ICSI cycles with fresh testicular spermatozoa carried out between January 1994 and December 2000 were analysed separately with regard to obstructive and non-obstructive azoospermia patients in this cohort follow-up study. Patients included in our study were from any social and ethnical group.

Female age was recorded on the first day of the initial ICSI cycle treatment. Only those cycles which reached the stage of oocyte retrieval and in which testicular sperm was used were included in the analysis.

Ovarian stimulation was performed using hMG. Down-regulation was carried out with GnRH agonist.

Assessment and handling of sperm/oocyte as well as the embryo transfer policy followed have been described extensively elsewhere (Van Steirteghem et al., 1996Go; Adonakis et al., 1997Go). For luteal-phase supplementation, micronized progesterone (600 mg per day) was administered intravaginally in three separate doses with or without additional hCG, three times 1500 IU, or 5000 IU single dose (Staessen et al., 1995Go).

The main outcome measure was delivery beyond 25 gestational weeks. Pregnancy follow-up was documented by our prospective follow-up programme for ICSI (Bonduelle et al., 1999Go).

The obstructive azoospermia group consisted of patients with congenital bilateral absence of vas deferens, post-infectious obstruction of vas deferens or failed vaso-vasostomy with normal spermatogenesis. The non-obstructive azoospermia group consisted of patients with histopathologically proven maturation arrest, atrophic sclerosis or germ cell aplasia (Sertoli cell-only syndrome). Klinefelter patients were excluded from the study. Patients with hypospermatogenesis were identified as a different clinical entity but were not included in the analysis of cumulative delivery rates in TESE–ICSI cycles, since the number of cycles performed was too small to permit meaningful conclusions.

Testicular sperm recovery
Open excisional testicular biopsies were performed under general or local anaesthesia. Experienced staff members of our institute performed the surgery. A 0.5–1 cm incision was made through the skin and the underlying layers. After incision of the tunica albuginea, gentle pressure was applied to the testicular mass and a small specimen (maximum 0.05 ml) of the testicular mass which protruded was removed by a pair of curved scissors. The testicular tissue was placed in a Petri dish containing HEPES-buffered modified Earle’s medium and transported to the adjacent laboratory. In the laboratory, the testicular tissue was teased apart with microscopic glass slides on the warmed stage of a stereomicroscope at x40 magnification. Under an inverted microscope (x400 magnification), the shredded tissue was then checked for the presence of spermatozoa. If no spermatozoa were observed, another biopsy specimen was taken. Surgery was terminated when spermatozoa were found or when the whole testicular mass had been bilaterally sampled at random. In obstructive azoospermia patients, the procedure was terminated after one biopsy while in non-obstructive azoospermia patients 5–10 biopsies were performed for each testis. During surgery, a randomly taken biopsy was sent for histopathological examination (Tournaye et al., 1997Go). Levin’s histopathological criteria were used for the classification of patients in different groups (Levin, 1979Go).

Statistical analysis
Cumulative delivery rates were estimated by life-table analysis using the Kaplan–Meier product limit procedure (Kaplan and Meier, 1958Go), and differences between groups were assessed by the log-rank test. Cumulative delivery rates were expressed as cumulative percentage probabilities with 95% confidence intervals (95% CIs). These computational procedures were run on SPSS for Windows version 10 (SPSS Inc., Chicago, IL, USA).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Testicular sperm recovery
Cumulative delivery rates after ICSI for obstructive and non-obstructive azoospermia patients are shown in Tables I and II respectively. Overall, the histopathological findings in 61% (364/599) of the patients were compatible with excretory duct obstruction as a cause of azoospermia, i.e. histopathology showed normal spermatogenesis. Conversely, 39% (235/599) of the patients were histopathologically compatible with secretory azoospermia.


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Table I. Cumulative delivery rates after ICSI in obstructive azoospermia patients with fresh TESE
 

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Table II. Cumulative delivery rates after ICSI in non-obstructive azoospermia patients with fresh TESE
 
Obstructive azoospermia
A total of 364 couples underwent a total of 609 fresh TESE–ICSI treatment cycles for obstructive azoospermia, leading to 141 deliveries. The mean age of the males was 39.3 years (95% CI, 38.4–40.2) while that of the females was 32.3 (95% CI, 31.8–32.8). The average number of cycles per patient was 1.7 (95% CI, 1.6–1.8) and the average number of cycles per patient required for a delivery was 4.3. The mean interval between consecutive TESE attempts was 9.6 months (95% CI, 8.3–10.9).

The cumulative delivery rate and life-table analysis for obstructive azoospermia are shown in Table I. The expected cumulative delivery rate increased in the first six cycles, after which it reached a plateau (79%).

Four triplets and 31 twin pregnancies were recorded. In addition two stillbirths occurred at term. No information could be obtained for six (1.6%) couples from abroad and these were assumed not to be pregnant for the purpose of this analysis.

Non-obstructive azoospermia
A total of 235 couples underwent 303 fresh TESE–ICSI treatment cycles, leading to 42 deliveries. The mean female age was 31.7 years (95% CI, 30.6–32.8), while that of the males undergoing testicular sperm recovery was 37.1 (95% CI, 35.6–38.6). The average number of cycles performed per patient was 1.3 and the average number of cycles required per patient for delivery was 7.2. The mean interval between consecutive TESE attempts was 9.4 months (95% CI, 7.3–11.5).

Life-table analysis overall and cumulative delivery rates according to histopathological classification in non-obstructive azoospermia are shown in Table II. The mean number of cycles required to achieve a delivery in patients with germ cell aplasia (Sertoli cell-only syndrome), those with maturation arrest and those with tubular sclerosis and atrophy were 5.8, 8.6 and 20 respectively. Four (1.7%) couples could not be contacted and these were assumed not to be pregnant for the purposes of this analysis.

The expected cumulative delivery rate increased in the first three cycles, after which it reached a plateau (31%).

The mean number of embryos replaced for obstructive azoospermia patients was 2.7 (range: 1–4) and for non-obstructive azoospermia patients was 2.8 (range: 1–4). Three triplets and eight twin pregnancies were recorded, while one stillbirth occurred at term.

A significant difference was apparent (P < 0.001) in the survival distributions observed in obstructive and non-obstructive azoospermia patients (Figure 1).



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Figure 1. Comparing expected cumulative delivery rates in patients with obstructive and non-obstructive azoospermia. The curve is shown as a solid line where data points represent outcomes involving adequate numbers of couples (~30) and as a dotted line where a lower number of couples renders the cumulative calculation less reliable. Data points are not presented where the number of couples falls below 10. This would result in the obstructive curve having five data points, and the non-obstructive curve having three data points.

 
Besides the study group, there were 38 patients with hypospermatogenesis and they underwent 51 cycles with a maximum of four cycles. Expected cumulative delivery rates reached a plateau at 30% after the second cycle and remained unchanged in the third and fourth cycles.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study shows that a lower expected cumulative delivery rate is to be anticipated in patients with non-obstructive azoospermia than for those with obstructive azoospermia following consecutive TESE–ICSI cycles. In patients with obstructive azoospermia, the expected cumulative delivery rate would be 48% after three cycles, while the corresponding figure in patients with non-obstructive azoospermia would be 31%.

Our data showed that after a few cycles, the delivery rate per cycle decreases compared with the first two cycles in patients with obstructive and non-obstructive azoospermia (Tables I and II). We observe the same trend in consecutive ICSI cycles with freshly ejaculated sperm (Osmanagaoglu et al., 1999Go, 2002). The delivery rate per cycle that is observed here is different from that in a recently published report (Shulman et al., 2002Go). The difference probably originates from not distinguishing between obstructive and non-obstructive azoo spermia and from the definition of non-obstructive azoospermia. However, expected cumulative pregnancy rates after five consecutive cycles are comparable.

In patients with non-obstructive azoospermia, a higher crude cumulative delivery rate after three TESE–ICSI cycles was observed for patients with Sertoli cell-only syndrome than for patients with maturation arrest and tubular sclerosis (22% versus 14 and 7%, respectively).

It has been shown that repetitive testicular biopsies within 3–6 months might decrease the possibility of sperm retrieval and harm testicular tissue due to permanent devascularization (Schlegel and Su, 1997Go; Westlander et al., 2001Go). In the current study, the mean time interval between consecutive TESE attempts was 9.6 and 9.4 months in patients with obstructive and non-obstructive azoospermia, respectively, and thus its potential influence on the results obtained is probably negligible.

A high dropout rate in couples with both non-obstructive and obstructive azoospermia was observed (Tables I and II), although this may be attributed to the fact that no cryopreserved TESE–ICSI cycles were included in the analysis. Only 8.5% (51/599) of the couples in the present study underwent both fresh and frozen TESE–ICSI cycles. Moreover, by including only patients in whom sperm was recovered, the dropout rate would be expected to be small. Probably the most important reason for discontinuing ICSI treatment was the fact that most of the patients were non-Belgian residents. In the beginning of the TESE–ICSI treatment era, they were referred to our centre for what was a novel treatment, but because of the rapid spread of the treatment worldwide, they continued their treatment in their home countries when no pregnancy ensued. Moreover, it is not possible to know whether these couples used the option of donor sperm since they could not be reached subsequently, rendering further follow-up inaccurate.

The other possible reasons for stopping ICSI treatment are: restricted numbers of cycles planned, treatment which is psychologically wearing, a financial cost that is too high, fertilization or cleavage problems, other medical problems, too advanced maternal age, divorce, death and other various reasons connected with work, relationships, transport or adoption (Osmanagaoglu et al., 1999Go). In our previous study (Osmanagaoglu et al., 1999Go), we included only Belgian residents and limited the time period of the initial cycles started, so as to ensure a long follow-up and a limited dropout. In the current study, all cycles with surgically retrieved testicular sperm were included without reference to residency, in order to have enough statistical power for the purposes of analysis.

The decision not to include those TESE–ICSI cycles in which sperm was cryopreserved in the life-table analysis was taken in order to avoid potential bias due to the fact that such cycles might be related to a deteriorating reproductive outcome (Palermo et al., 1999Go).

It is expected that the wider application of TESE–ICSI will allow a larger population of patients to be analysed and thus lead to conclusions for specific subgroups of male patients and/or extend the analysis to cover several parameters such as female age, female diagnosis, number of oocytes retrieved and embryos yielded, and the method of sperm retrieval.

At present, however, couples in whom the male partner is diagnosed with obstructive azoospermia may be counselled that their chances of achieving delivery within three ICSI cycles with freshly retrieved testicular sperm are 48%. In addition, couples with non-obstructive azoospermia have an expected delivery rate after two cycles of 19% and after three cycles of 31%.


    Acknowledgements
 
The authors wish to thank the clinical, scientific, nursing and technical staff of the Center for Reproductive Medicine especially the nurses and technicians involved in the follow-up project. Mr Frank Winter of the Language Education Center at our University corrected the manuscript. This work was supported by grants from the Fund for Scientific Research-Flanders.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on June 12, 2002; accepted on May 9, 2003.