The results of 154 ICSI cycles using surgically retrieved sperm from azoospermic men

J.D.M. Nicopoullos1,3, C. Gilling-Smith1, P.A. Almeida1 and J.W.A. Ramsay2

1 Assisted Conception Unit, Chelsea & Westminster Hospital, London SW10 9NH and 2 Department of Urology, Charring Cross Hospital, London W6 8RF, UK

3 To whom correspondence should be addressed. e-mail: James.nicopoullos{at}chelwest.nhs.uk


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: The effects of source of sperm, aetiology and sperm cryopreservation on ICSI cycles in azoospermic men were evaluated. The effect of aetiology of azoospermia on embryo development was also assessed. METHODS: This study was a retrospective analysis of 154 cycles (91 couples) using surgically retrieved sperm. Outcome measures were fertilization rate (FR), implantation rate (IR), and clinical pregnancy rate (CPR) and livebirth rate (LBR) per transfer. RESULTS: Our data demonstrated similar outcome between the use of epididymal or testicular sperm in men with obstructive azoospermic (OA). FR and IR were significantly lower (P < 0.05) using sperm from men with non-obstructive azoospermic (NOA), but although pregnancy outcome appeared lower, this did not reach statistical significance (P = 0.08). Cryopreservation of epididiymal sperm did not alter outcome, but the use of frozen–thawed testicular sperm did demonstrate a lower FR, with no statistical difference in IR or pregnancy outcome. Embryos derived from NOA sperm had impaired development beyond day 2 post-oocyte retrieval (OA, 44% <5 cell; NOA, 71% <5 cell; P = 0.002). CONCLUSIONS: The use of sperm from men with NOA significantly affects fertilization and implantation in ICSI cycles. The use of frozen–thawed testicular sperm affects fertilization rate without significantly altering pregnancy outcome. The use of such data on which to base clinical decisions needs to be supported by the meta-analyses of previous reports.

Key words: azoospermia/ICSI/male factor subfertility/surgical sperm retrieval


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Ten years ago, the use of donor sperm was the only option offering a realistic chance of conception for the azoospermic or severely oligoasthenoteratospermic male. The introduction of ICSI by the work of Van Steirteghem and colleagues in Brussels revolutionized the management of male factor fertility. The same authors demonstrated high fertilization rates (FRs) and implantation rates (IRs) with ICSI in a series of 150 consecutive treatment cycles in couples previously not accepted for IVF or who had failed fertilization with conventional IVF (Van Steirteghem et al., 1993Go). The ability of ICSI to achieve high fertilization and pregnancy rates, regardless of semen parameters (Nagy et al., 1995aGo), led to its application in azoospermic patients.

The majority of series of ICSI cycles from azoospermic men, using both fresh and frozen–thawed sperm and from men with both obstructive (OA) and non-obstructive (NOA) azoospermia have reported acceptable fertilization and pregnancy outcome. However, debate remains about the most suitable site for retrieval and the role of freezing.

Reports comparing ICSI cycles using epididymal and testicular sperm in OA patients have all found similar outcomes (Hovatta et al., 1995Go; Nagy et al., 1995bGo; Silber et al., 1995aGo,bGo; Fahmy et al., 1997Go; Mansour et al., 1997Go; Rosenlund et al., 1997Go; Dohle et al., 1998Go; Palermo et al., 1999Go). However, the data comparing ICSI outcomes between patients with OA and NOA is less consistent. Although some suggest no difference (Devroey et al., 1996Go; Windt et al., 2002Go), the majority of reports show significantly impaired fertilization or pregnancy outcome in cycles using testicular sperm from NOA patients compared with testicular sperm from OA patients (Kahraman et al., 1996Go; Fahmy et al., 1997Go; Mansour et al., 1997Go; Palermo et al., 1999Go; De Croo et al., 2000Go; Pasqualotto et al., 2002Go).

Similarly, the outcome of cycles using fresh or frozen–thawed retrieved spermatozoa is the source of much debate. The majority or reports suggest no significant worsening in outcome with the use of cryopreserved gametes after surgical retrieval (Devroey et al., 1995Go; Nagy et al., 1995bGo; Silber et al., 1995bGo; Friedler et al., 1997Go; 1998Go; Tournaye et al., 1999Go; Habermann, 2000Go; Sousa et al., 2002Go; Windt et al., 2002Go), including many that compared fresh and frozen cycles in the same patients after the same sperm retrieval (Gil-Salom et al., 1996Go; Oates et al., 1996Go; Cayan et al., 2001Go). Others, however, have reported a significantly lower FR (De Croo et al., 1998Go; Wood et al., 2002Go), clinical pregnancy rate (CPR) (Palermo et al., 1999Go; Christodoulou et al., 2002Go) and IR (De Croo et al., 1998Go; Christodoulou et al., 2002Go) using cryopreserved spermatozoa. The numbers in many of the reported studies have been small, and only one (Palermo et al., 1999Go) has included over 150 cycles to compare ICSI outcome by source of sperm, aetiology of azoospermia, and by the use of cryopreserved sperm.

Reports have also indicated an influence on embryo development of paternal factors. The use of ICSI compared with IVF (Dumoulin et al., 2000Go; Miller and Smith, 2001Go) has been shown to lower blastocyst formation. Semen parameters and aetiology of azoospermia have also been shown to correlate with blastulation rate and quality of resulting blastocysts (Balaban et al., 2001Go; Miller and Smith, 2001Go).

The aim of our study is to present our data on ICSI cycles using surgically retrieved sperm from azoospermic men, assess embryo development and compare the outcomes of ICSI for: (i) epididymal versus testicular sperm; (ii) fresh versus frozen–thawed sperm; and (iii) OA versus NOA.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This retrospective analysis involved 154 consecutive ICSI cycles performed between November 1996 and July 2002 (from 91 couples) using fresh or cryopreserved surgically retrieved sperm in azoospermic patients.

Prior to assisted reproduction, the 91 men were evaluated to determine the aetiology of azoospermia by medical history, examination and hormonal assay, karyotype and, where appropriate, cystic fibrosis gene mutation analysis. The female partners were also assessed with full history and infertility investigation, and any factors classified according to the Hull and Rutherford classification of infertility (Rutherford and Jenkins, 2002Go).

The aetiology of the azoospermia was classified as OA (127 cycles) or NOA (25 cycles) based on history, examination, male FSH levels and surgical findings. In two cycles, insufficient information was obtained to allow accurate classification of aetiology, and they were therefore excluded from the analysis of the data by aetiology. The aetiology of the azoospermia in our patient group can be seen in more detail in Table I.


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Table I. Aetiology of azoospermia in patients undergoing ICSI cycles
 
Sperm retrieval and assisted reproduction
The data presented in this study were based on clinical results obtained from four assisted reproductive centres. In all cases, surgical sperm retrieval was performed by the same surgeon. Epididymal sperm was retrieved by microsurgical sperm aspiration (MESA) under general anaesthesia. The samples were prepared in the IVF laboratory by washing in 1–2 ml of culture medium. The suspension was centrifuged at 200 g for 10 min and the pellet redispersed in 50–100 µl of culture medium. Presence of motile sperm was assessed under an inverted microscope (x400 magnification, Hoffman modulation). The prepared samples were then left in the incubator, set at 37°C in an atmosphere of 5% CO2 in air, until use. In cases where no motile sperm were found in the MESA sample (by an embryologist present at the surgical procedure), a testicular biopsy was performed (TESE). The tubules were minced using two insulin needles and sterile glass slides, and sperm suspension was prepared as described above. Epididymal or testicular sperm suspension was pipetted into microdrops of culture medium under paraffin oil. Under the inverted microscope, a motile sperm was immobilized using a microinjecting pipette and placed into a drop of polyvinylpyrolidone (MediCult, Redhill, UK) prior to ICSI.

Surplus spermatozoa were cryopreserved for future ICSI attempts, thus avoiding the need for repeated microsurgery. The sperm suspension was diluted with equal volume of sperm cryopreservation medium (MediCult), aliquoted into cryovials and suspended in liquid nitrogen vapour for 30 min before being submerged in liquid nitrogen for storage. On the morning of oocyte retrieval, a vial of frozen sample was thawed at room temperature for 20 min. Samples were subsequently prepared as described earlier.

Oocyte retrieval was performed after pituitary desensitization with GnRH agonists (long protocol) and ovulation stimulation with gonadotrophins. The oocytes were stripped of surrounding cumulus cells using hyaluronidase (MediCult) and ICSI performed between 38–42 h post-HCG injection. Fertilization was assessed 16–18 h post-ICSI, and the most developmentally advanced and morphologically normal embryos were subsequently transferred on either day 2 (cleaved to the 2- to 4-cell stage by 42–49 h post-injection) or day 3 (cleaved to the 5- to 8-cell stage by 64–72 h post-injection) post-oocyte retrieval. The embryos were graded according to size and shape of blastomeres, and degree of fragmentation (grade 1, evenly sized blastomeres with no fragmentation; grade 2, evenly sized blastomeres with moderate degree of cytoplasmic fragmentation of 25%; grade 3, uneven or indistinct blastomeres with significant fragmentation of >25%). Luteal support was administered in the form of progesterone pessaries (800 mg/day; Cyclogest®; Shire Pharmaceuticals, Basingstoke, UK) from the day of transfer and continued until the 12th week of pregnancy, where appropriate.

Pregnancy was detected using urinary or serum {beta}-HCG tests and clinical pregnancy determined by observation of a gestational sac at a 6-week ultrasound scan. Implantation rate was defined as the number of gestation sacs observed divided by the number of embryos transferred.

Statistical analysis was performed using Fisher’s exact test and {chi}2-test.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Epididymal versus testicular cycles
A total of 127 cycles were performed using epididymal sperm and 27 using testicular sperm. Patient characteristics and clinical outcome of these cycles are shown in Table II. The only variable that was statistically different between the two groups was paternal age (40.6 versus 35.9 years, respectively; P = 0.002). This was largely due to the epididymal group including a large number of post-vasectomy men (mean vasectomy treatment interval of 15 years). Factors with potential impact on outcome, such as maternal age (33.2 versus 32.6 years), BMI (23.8 versus 20.7 kg/m2), maternal serum FSH (7.1 versus 7.9 IU/l), total dose of FSH required (2760 versus 2703 IU) and the proportion of women with fertility issues that could affect outcome (endometriosis, fibroids, tubal pathology, polycystic ovaries, etc.) (46 versus 40%), were not significantly different between the groups. The number of eggs collected (13.1 versus 11.2) and FR (48.6 and 45.6%) were also not significantly different. The percentage of cycles not reaching embryo transfer was higher in the testicular sperm group, and this discrepancy was magnified if the cycles cancelled due to maternal-related factors (i.e. poor response to stimulation) were excluded (11.1% testicular versus 4.7% epididymal).


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Table II. Comparison of ICSI cycles using epidiymal and testicular sperm
 
The number of embryos transferred per cycle was similar between the two groups, with a higher proportion of top quality embryos and lower proportion of poor quality embryos transferred in the epididymal group. Analysis of the outcomes showed a non-significant increase in FR (49 and 46%), IR (13 and 11%), CPR (25 and 22%) and live-birth rate (LBR) (18 and 13%) using epididymal sperm compared with testicular sperm.

To eliminate the potential effects of aetiology of azoospermia in the two groups, we also compared epididymal (n = 113) and testicular cycles (n = 14) in men with OA only. When only these cycles were analysed, the clinical outcomes were comparable, with no statistically significant differences in the parameters examined (epididymal: FR 49%, IR 15%, CPR 28% and LBR 20%; testicular: FR 51%, IR 15%, CPR 29% and LBR 21%). Furthermore, there were no cycles cancelled in the testicular group.

OA versus NOA cycles
A total of 127 and 25 cycles were performed using sperm from men with OA and NOA, respectively (Table III). Again, paternal age was higher in the OA group as a consequence of the post-vasectomy patients (OA versus NOA, 40.9 versus 34.4 years; P = 0.006). The total dose of gonadotrophins required for stimulation was higher in the NOA group, despite similar maternal age (3505 versus 2604 IU). Also of note was that a lower proportion of women in the NOA group had factors that might impair fertility (Hull criteria: 52% OA versus 12% NOA). The FR of 49.1% in the OA group was significantly higher than the 42.1% in the cycles from NOA patients (P = 0.05). The quality of resulting embryos transferred was better in the OA group compared with the NOA group (grade 1, 34.7 versus 22.8%; grade 3 or worse, 8.5 versus 19.3%). Thus, the mean number of embryos transferred was higher in NOA men (2.59 versus 2.38: P < 0.05). Analysis of outcome showed a significantly lower FR (49.1 and 42.1%; P = 0.05) and IR (15.2 and 3.5%; P = 0.02) using sperm from men with NOA compared with OA. The CPR (28.1 and 9.1%; P = 0.06) and LBR (20.2 and 4.5%; P = 0.08) were also lower, but these findings were of borderline statistical significance (P < 0.1). There was no difference in the miscarriage rate between the two groups.


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Table III. Comparison of ICSI cycles in men with OA and NOA
 
Fresh versus frozen sperm cycles
In all, 64 and 90 cycles were performed using fresh and frozen sperm respectively (Table IV). The patient characteristics were similar, as were the number of eggs collected per cycle (12.0 versus 13.3). Analysis of outcome showed no significant difference in FR (48 versus 48%), IR (15 versus 12%), CPR (23 versus 26%) or LBR (18 versus 17%) using fresh sperm and frozen–thawed sperm.


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Table IV. Comparison of ICSI cycles using fresh and frozen sperm
 
When these cycles are further broken down by source of sperm, the FR, IR and pregnancy outcomes rate remain unaffected by cryopreservation of epididymal sperm, with no significant difference in any outcome measure. However, cycles using testicular sperm demonstrated a higher FR using fresh sperm compared with frozen (50 versus 39%), of borderline statistical significance (P < 0.1), with similar CPR (23 versus 20%) and LBR (15 versus 10%). Although, the IR was lower (14 versus 9%) and miscarriage rate higher (66 versus 33%) when using frozen testicular sperm, these findings did not reach statistical significance.

The effect of the aetiology of azoospermia on cryopreservation was also analysed. The FR was unaffected by cryopreservation in both OA (49.5 and 48.8%) and NOA (43.5 versus 39.8%). The IR, CPR per transfer and LBR per transfer were 17.9, 26.1 and 21.7%, respectively, for cycles using fresh sperm from OA men; 13.6, 29.4 and 19.1% using frozen sperm from OA men; 5, 11.1 and 0% using fresh sperm from NOA men; and 2.7, 7.7 and 7.7% using frozen sperm from NOA men. All outcomes were not significantly different between fresh and frozen cycles in the OA group. Statistical analysis was not performed between the NOA groups due to the small numbers in each.

A small subgroup (14 patients, 12 with OA and two with NOA) completed cycles with both fresh and frozen sperm from the same retrievals (Table V). Maternal age, number of eggs collected and proportion of mature eggs collected for injection was similar between the two groups. However, the total dose of gonadotrophin used for stimulation was higher in the cycles using frozen sperm. The data showed that FR, IR, and CPR and LBR per transfer were not impaired with cryopreservation of retrieved gametes in this subgroup.


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Table V. Comparison of fresh and frozen sperm cycles in the same patients from the same retrieval
 
Embryo development
The development of embryos available for transfer was assessed on the day of transfer (Table VI). There was no significant difference in the proportion of embryos that had reached the 2- to 3-cell stage (42%) and the 4-cell stage (48.0 versus 42.2%) by day 2 when comparing cycles in OA and NOA, respectively. However, when embryos available for transfer were assessed on day 3, a significantly higher proportion had arrested, i.e. ≤4-cells (71.1%) when derived from sperm from men with NOA compared with OA (44.0%; P = 0.002).


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Table VI. Comparison of embryo development by source of sperm
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The use of surgical sperm retrieval and ICSI has revolutionized the treatment of azoospermic men, but there is still a lack of concensus on the effect of source of sperm, aetiology of azoospermia and cryopreservation.

The results presented here show an equal outcome using epididymal or testicular sperm in patients with similar aetiology (Table II). To avoid possible bias from the use of subsequent cycles from the same patient, we also analysed only the first cycle for each couple. There remained no significant difference in pregnancy outcome (CPR 24 versus 22%). This is in agreement with other large retrospective analyses (Fahmy et al., 1997Go; Palermo et al., 1999Go). Therefore, where motile sperm are not readily found at epididymal aspiration in a man with OA, the use of sperm retrieved at testicular biopsy is an acceptable alternative. Where a diagnosis of OA is made, however, epididymal aspiration remains the method of choice to minimize the potential consequences of testicular retrieval of inflammation, haematoma formation and devascularization (Schlegel and Su, 1997Go).

Although simple sperm aspiration procedures such as percutaneous epididymal sperm aspiration (PESA) and testicular epididymal sperm aspiration (TESA) are as effective as an open MESA in terms of retrieval rates, ICSI outcome (Tournaye et al., 1998Go) and patient satisfaction (Gorgy et al., 1998Go), and do not necessarily require the expertise of a urological surgeon, we still recommend the use of MESA as the first-line method of retrieval in men with OA in view of the opportunity it gives for diagnosis, reconstruction where appropriate, and retrieval of greater numbers of sperm for cryopreservation. Furthermore, the use of blind procedures such as PESA may result in more diffuse injury to epididymal tubules with subsequent scarring. This has been suggested to impair the efficacy of repeat procedures often required in view of the lower numbers of sperm retrieved (Pasqualotto et al., 2003Go).

Kahraman et al. (1996)Go was the first to report a significantly lower FR (65 versus 34%) and CPR (62 versus 42%) using sperm from NOA men. This was later confirmed by larger studies (Fahmy et al., 1997Go; Mansour et al., 1997Go). Some have reported impaired fertilization but similar pregnancy outcome in NOA (Palermo et al., 1999Go; De Croo et al., 2000Go), and others similar fertilization but impaired pregnancy outcome (Pasqualotto et al., 2002Go). The most recent report showed no impairment in any outcome measure (Windt et al., 2002Go). Our findings confirm the majority of previous reports, with significantly impaired FR and IR(P < 0.05), and impaired pregnancy outcome of borderline clinical significance (P < 0.10). If however, only the first cycle from each patient is analysed, CPR is then significantly worse in men with NOA compared with OA (0 versus 22%; P < 0.05), although the numbers in the latter group are small. The inconsistency between the published data may be due to several factors, such as differing diagnostic criteria and types of retrieval. We therefore used strict diagnostic criteria and only MESA or TESA in all patients.

Our data show a significantly lower FR when testicular sperm is used frozen–thawed (50 versus 39%). This is in agreement with previous reports from De Croo et al. (1998)Go (79 versus 71%) and Wood et al. (2002)Go (71 versus 53%), with no effect of cryopreservation seen on epididymal sperm. Although the pregnancy rates in this report are not significantly impaired by cryopreservation of retrieved sperm, the IR shows a trend suggestive of impaired implantation following the use of with frozen–thawed samples, which does not reach clinical significance but does mirror previous reports (De Croo et al., 1998Go; Tournaye et al., 1999Go; Habermann et al., 2000Go). Furthermore, the number of embryos transferred per cycle in our frozen sperm groups is higher.

The majority of reports comparing fresh versus frozen testicular sperm (including our own) use sperm from a combination of men with OA and NOA, without comparing the effect of cryopreservation by aetiology of infertility. This is predominantly due to the low numbers that would be analysed in each group. If we compare fresh and frozen testicular sperm in our OA patients only (n = 14), we confirm our finding of a statistically significant difference in FR (56.4 versus 45.1% respectively), but numbers are too small for meaningful analysis of pregnancy and implantation outcome. Only three papers have compared fresh and frozen–thawed testicular sperm from only men with NOA (Friedler et al., 1997Go; 2002Go; Sousa et al., 2002Go), and these showed no significant differences in either fertilization or pregnancy outcome. Meta-analysis of published data, including our own, would improve our ability to make clinical decisions based on these findings (Nicopoullos et al., 2003Go).

The findings in the subgroup of patients who had both a fresh and frozen cycle (Table V) are similar to previous reports that showed similar pregnancy outcomes (Gil-Salom et al., 1996Go; Oates et al., 1996Go; Ben-Yosef et al., 1999Go; Cayan et al., 2001Go; Wood et al., 2002Go). The improved outcome using frozen sperm in this group may be explained by the experience of the initial cycle leading to an improved stimulation regime, therefore optimizing the number of mature oocytes and improving fertilization outcome. This is supported by the increased total dose of gonadotrophins used in the frozen sperm group. These findings are further supported by a recent report of improved outcome using cryopreserved compared with fresh epidiymal sperm in men with OA (Wood et al., 2003Go).

Lower blastulation rate has been demonstrated in ICSI compared with IVF embryos, regardless of culture medium used or conditions (Dumoulin et al., 2000Go). Miller and Smith (2001)Go, who also reported similar findings, assessed the influence of paternal factors, and found a significant correlation between progressive motility and sperm morphology and blastocyst development and quality. Thereafter, Balaban et al. (2001)Go found that embryos generated using spermatozoa in non-obstructive cases, when used for ICSI, resulted in a lower blastulation and IR than embryos generated from ejaculated sperm or sperm from OA men. These studies suggest a putative paternal influence on embryo development and implantation. Even with normal peripheral karyotyping, sperm from men with NOA have been shown to have a higher incidence of chromosomal abnormality using fluorescence in situ hybridization analysis (Palermo et al., 2002Go). Our data support these findings. By day 2 post-egg collection, embryos from ICSI cycles injected with sperm from men with NOA were developing at a similar rate to those derived from cycles using sperm from men with OA. However, by day 3 the proportion of embryos the development of which had arrested was significantly higher in the NOA couples. This finding supports a sperm-derived influence on embryo development through the activation of the embryonic genome that occurs at day 3 of embryogenesis, i.e. at the 4- to 8-cell stage (Braude et al., 1988Go).

The overall results of our series of sperm retrieval cycles (FR 48%, IR 13%, CPR 25%, LBR 18%) clearly demonstrate acceptable success for azoospermic men undergoing ICSI, although overall, outcome is lower compared with all ICSI cycles (FR 57%, IR 16%, CPR 31%, LBR 29%) during the same period of analysis, regardless of source of sperm, in our unit.

In summary, our data confirm a similar outcome in men with OA using epididymal or testicular sperm, with significantly impaired outcome in cycles using sperm from men with NOA. Implantation is also impaired using frozen–thawed testicular sperm, with no effect on pregnancy outcome. The use of such data on which to base clinical decisions needs to be supported by meta-analyses of previous reports.


    Acknowledgements
 
We greatly appreciate the input of nursing, clinical and embryological staff during the treatment of these patients. We would also like to thank Mr Ian Grace of Imperial College, London, for his statistical analyses.


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 Materials and methods
 Results
 Discussion
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Submitted on June 20, 2003; resubmitted on September 5, 2003; accepted on October 23, 2003.