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
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Abstract |
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Key words: azoospermia/ICSI/male factor subfertility/surgical sperm retrieval
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Introduction |
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The majority of series of ICSI cycles from azoospermic men, using both fresh and frozenthawed 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., 1995; Nagy et al., 1995b
; Silber et al., 1995a
,b
; Fahmy et al., 1997
; Mansour et al., 1997
; Rosenlund et al., 1997
; Dohle et al., 1998
; Palermo et al., 1999
). However, the data comparing ICSI outcomes between patients with OA and NOA is less consistent. Although some suggest no difference (Devroey et al., 1996
; Windt et al., 2002
), 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., 1996
; Fahmy et al., 1997
; Mansour et al., 1997
; Palermo et al., 1999
; De Croo et al., 2000
; Pasqualotto et al., 2002
).
Similarly, the outcome of cycles using fresh or frozenthawed 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., 1995; Nagy et al., 1995b
; Silber et al., 1995b
; Friedler et al., 1997
; 1998
; Tournaye et al., 1999
; Habermann, 2000
; Sousa et al., 2002
; Windt et al., 2002
), including many that compared fresh and frozen cycles in the same patients after the same sperm retrieval (Gil-Salom et al., 1996
; Oates et al., 1996
; Cayan et al., 2001
). Others, however, have reported a significantly lower FR (De Croo et al., 1998
; Wood et al., 2002
), clinical pregnancy rate (CPR) (Palermo et al., 1999
; Christodoulou et al., 2002
) and IR (De Croo et al., 1998
; Christodoulou et al., 2002
) using cryopreserved spermatozoa. The numbers in many of the reported studies have been small, and only one (Palermo et al., 1999
) 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., 2000; Miller and Smith, 2001
) 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., 2001
; Miller and Smith, 2001
).
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 frozenthawed sperm; and (iii) OA versus NOA.
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Materials and methods |
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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, 2002).
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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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 3842 h post-HCG injection. Fertilization was assessed 1618 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 4249 h post-injection) or day 3 (cleaved to the 5- to 8-cell stage by 6472 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 -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 Fishers exact test and 2-test.
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Results |
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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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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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Discussion |
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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., 1997; Palermo et al., 1999
). 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, 1997
).
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., 1998) and patient satisfaction (Gorgy et al., 1998
), 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., 2003
).
Kahraman et al. (1996) 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., 1997
; Mansour et al., 1997
). Some have reported impaired fertilization but similar pregnancy outcome in NOA (Palermo et al., 1999
; De Croo et al., 2000
), and others similar fertilization but impaired pregnancy outcome (Pasqualotto et al., 2002
). The most recent report showed no impairment in any outcome measure (Windt et al., 2002
). 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 frozenthawed (50 versus 39%). This is in agreement with previous reports from De Croo et al. (1998) (79 versus 71%) and Wood et al. (2002)
(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 frozenthawed samples, which does not reach clinical significance but does mirror previous reports (De Croo et al., 1998
; Tournaye et al., 1999
; Habermann et al., 2000
). 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 frozenthawed testicular sperm from only men with NOA (Friedler et al., 1997; 2002
; Sousa et al., 2002
), 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., 2003
).
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., 1996; Oates et al., 1996
; Ben-Yosef et al., 1999
; Cayan et al., 2001
; Wood et al., 2002
). 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., 2003
).
Lower blastulation rate has been demonstrated in ICSI compared with IVF embryos, regardless of culture medium used or conditions (Dumoulin et al., 2000). Miller and Smith (2001)
, 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)
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., 2002
). 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., 1988
).
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 frozenthawed 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.
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Acknowledgements |
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Submitted on June 20, 2003; resubmitted on September 5, 2003; accepted on October 23, 2003.