1 Departments of Urology and 2 Obstetrics and Gynecology and Reproductive Sciences, University of California San Francisco School of Medicine, San Francisco, CA, USA
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Abstract |
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Key words: cryopreservation/epididymal spermatozoa/ICSI/IVF
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Introduction |
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Given this success, several centres initiated the use of frozen-thawed epididymal spermatozoa in addition to freshly retrieved spermatozoa to achieve ICSI pregnancies. The apparent clinical benefits inherent in epididymal sperm cryopreservation were immediately obvious to both patients and providers: the use of frozenthawed epididymal spermatozoa could reduce the number of male partner procedures required for pregnancy (Oates et al., 1996; Nudell et al., 1998
) as well as minimize the inconvenience associated with sperm retrieval performed concurrently with ovulation induction (Oates et al., 1996
). Furthermore, data that address the cryobiological behaviour of epididymal spermatozoa have demonstrated that it is quite feasible to consider frozen-thawed epididymal spermatozoa for ICSI procedures (Bachtell et al., 1999
).
The most important issue on this matter, however, is whether frozenthawed epididymal spermatozoa provide clinical outcomes equivalent to that of fresh spermatozoa. To date, the published experience with frozenthawed epididymal spermatozoa and ICSI suggests that fertilization and pregnancy success rates are comparable to those achieved with freshly retrieved spermatozoa (Nagy et al., 1995; Oates et al., 1996
; Cha et al., 1997
; Holden et al., 1997
; Friedler et al., 1998
; Hutchon et al., 1998
; Van Steirteghem et al., 1998
; Tournaye et al., 1999
). However, the majority of studies use historical or unmatched control patients to compare clinical outcomes. Three studies reported clinical outcomes in patients who underwent at least two IVF/ICSI cycles, one with fresh and a subsequent cycle with frozenthawed epididymal spermatozoa (Friedler et al., 1998
; Hutchon et al., 1998
; Tournaye et al., 1999
). These studies represent a more powerful design because female factor variables were better controlled. Despite better design, the aims of these studies varied from a comparison of results with different epididymal sperm retrieval techniques (Friedler et al., 1998
) to an examination of clinical outcomes in couples who had subsequent but not necessarily consecutive IVF/ICSI cycles (Hutchon et al., 1998
). To further clarify this issue, we sought to assess ICSI outcomes in couples each of whom underwent consecutive IVF/ICSI cycles, the first with fresh and the second with frozenthawed epididymal spermatozoa obtained from a single aspiration procedure.
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Materials and methods |
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Microsurgical sperm aspiration procedure (mini-MESA)
Epididymal sperm aspiration was performed in all patients by a single surgeon (P.J.T.) as previously described (Nudell et al., 1998). Briefly, under local anaesthesia, a small scrotal incision was made and the epididymis exposed lateral to the testis. The epididymal tunic was incised over a dilated epididymal tubule using microsurgical technique. Individual epididymal tubules were entered with microscissors and fluid with spermatozoa aspirated into small syringes and subsequently into sterile 5 ml test tubes (Becton Dickinson, Franklin Lakes, NJ, USA) containing Earle's Medium (Gibco BRL, Grand Island, NY, USA) supplemented with 4 mmol/l sodium bicarbonate, 21 mmol/l HEPES (Calbiochem-Novabiochem Corp., La Jolla, CA, USA), 0.47 mmol/l pyruvate, and 10% v/v synthetic serum substitute (SSS; Irving Scientific, Santa Ana, CA, USA) and maintained at 37°C. At each epididymal site, 10 µl of extracted fluid was examined under x400 bright-field microscopy for the presence of motile spermatozoa. Epididymal sites chosen for aspiration progressed from the distal (cauda) to proximal (caput) epididymis until motile spermatozoa were observed in the aspirate. The epididymal tubules and tunic were closed with interrupted 100 and 90 nylon sutures respectively. Aspirated epididymal fluid was immediately transferred to the IVF laboratory for micromanipulation and processing.
Oocyte stimulation and retrieval, ICSI and embryo transfer
As described previously, female partners underwent pituitary desensitization with a gonadotrophin hormone-releasing agonist and ovarian stimulation with human menopausal gonadotrophins (Pergonal or Fertinex; Serono, Norwell, MA, USA) (Nudell et al., 1998). Retrieved oocytes were handled in the laboratory according to the technique of Hillier et al. (1984) and cultured in Earle's medium with 5% CO2 at 37°C. After 2 h incubation in medium, oocytes were denuded of cumulus cells using 80 U/ml hyaluronidase (Sigma Chemical Co., St Louis, MO, USA).
Motile epididymal spermatozoa were minimally processed and handled prior to micromanipulation. For ICSI, morphologically normal and progressively motile spermatozoa were recovered using a `swim-out' technique (Conaghan et al., 1997; Nudell et al., 1998
). Briefly, 5 µl of undiluted sperm suspension was pipetted to the centre of a microdroplet (7 µl) of polyvinylpyrrolidine (PVP; Irvine Scientific). Spermatozoa reaching the microdroplet edge were collected with a finely drawn ICSI pipette (Pacific Andrology Inc., Montrose, CA, USA), transferred to a clean droplet of PVP for tail breaking, and injected into oocytes.
Following ICSI, oocytes were transferred into culture medium under mineral oil (Sigma) and incubated for 16 h. Normal fertilization was confirmed when two pronuclei were clearly visible in the oocyte cytoplasm. Embryos were transferred to the uterus 24 or 48 h after fertilization. Successful implantation was determined by appropriately rising serum ß-HCG concentrations. A clinical pregnancy was confirmed with the presence of a fetal sac and a heart beat on vaginal ultrasound 46 weeks later.
Sperm analysis and cryopreservation and thaw protocol
Aspirated spermatozoa not used for ICSI during the first IVF cycle were prepared for analysis and cryopreservation. Sperm concentration and motility analyses were performed on both fresh and frozenthawed spermatozoa from each patient according to World Health Organization criteria (WHO, 1992) under light microscopy at 37°C after dilution and preparation on a microcell slide (Conception Technologies, San Diego, CA, USA). Motility progression was scored as follows: 0 = immotile spermatozoa; 1 = non-progressive, twitching spermatozoa; 2 = slow progressive or sluggish motility; 3 = rapid forward progressive motility; 4 = capacitated (hyperactivated) progression (Mortimer, 1994
). In addition, the total motile sperm count (TMC: volumexconcentrationxmotile fraction) was determined for the entire retrieved specimen from each patient.
Aspirated spermatozoa to be cryopreserved were washed twice (centrifuged at 170 g, 10 min) in phosphate-buffered saline (Gibco) with 2% v/v SSS. The final pellet was resuspended in 0.5 ml of Earle's medium. Specimens were cryopreserved in a 1:1 dilution of sperm suspension to test yolk buffer (Irvine Scientific) in 1.0 ml vials by the slow freeze method (Mahadevan and Trounson, 1983) in a Planar Biomed controlled rate freezer (T.S. Scientific, Perkasie, PA, USA). Upon thawing for the subsequent IVF cycle, vials were thawed by incubation at 37°C for 10 min. The thawed spermatozoa were then gradually reintroduced into isotonic medium by adding an equal volume of Earle's medium to the vial over a 5 min period. This diluted specimen was centrifuged (170 g, 10 min). This dilution and wash procedure was repeated twice and the final pellet resuspended in 0.5 ml of Earle's buffered medium for both ICSI and reassessment of sperm parameters. Motile spermatozoa were used for all ICSI procedures in this study.
Assessment of embryo quality
Embryos derived from each IVF/ICSI cycle were assessed for quality according to an established grading system prior to embryo transfer. Embryos were observed under a zoom stereomicroscope (model SZH-ILLB: Olympus Optical Co. Ltd, Japan), at a magnification of x128, illuminated using a 6 V, 20 W halogen bulb. Embryo quality was assessed by a single embryologist (J.C.) at 72 h after retrieval of oocytes and rated on a scale of 1 = excellent to 5 = poor (Plachot et al., 1986; Scott et al., 1991
).
Statistical analysis
Statistical analysis was performed using the paired t-test to compare fresh and frozenthawed sperm parameters and the 2-test to compare ICSI outcomes. P < 0.05 was considered significant.
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Results |
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The mean age of the male partners at the time of epididymal sperm retrieval was 45.9 ± 12.9 years (range 2971). The causes of obstruction were congenital bilateral absence of the vas deferens (CBAVD) in 10 patients (53%), vasectomy or failed vasectomy reversal in seven patients (37%) and idiopathic epididymal obstruction in two patients (10%). Epididymal sperm retrieval was successful in all cases in which it was attempted.
Fresh and frozenthawed epididymal sperm parameters
Epididymal sperm parameters before and after cryopreservation are given in Table I. As expected, there was a significant difference in sperm motility when the fresh and frozenthawed epididymal spermatozoa were compared (P = 0.004). However, no significant differences were observed in forward progression of motility between the two specimens.
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Discussion |
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A summary of the ICSI experience with fresh and frozenthawed epididymal spermatozoa is given in Table III. Most of the prior studies on this issue generally lack control groups or use controls derived from historical series or parallel but different groups of patients. In a study that reviewed a large early experience with epididymal spermatozoa and ICSI, Van Steirteghem et al. (1998) reported higher fertilization (64.8% and 54.7%), and pregnancy rates (44.3 and 33.8%) with fresh versus frozenthawed epididymal spermatozoa. Conversely, Shibahara et al. (1999) reported very significant differences in fertilization rates (68.6 versus 45.3%) and pregnancy rates (60 versus 23.1%) when fresh and frozenthawed epididymal spermatozoa were compared among different couples. Importantly, however, in cases in which only motile spermatozoa were used for ICSI, oocyte fertilization rates between fresh (68.6%) and frozenthawed (68.4%) epididymal spermatozoa were virtually identical. In general, oocyte fertilization rates after ICSI with frozenthawed epididymal spermatozoa ranged from 37 to 72%, which compares favourably to that found with fresh epididymal spermatozoa (range 2073%). Thus, this body of work suggests that excellent ICSI fertilization rates are achievable with frozen and thawed epididymal spermatozoa.
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In the current study, we selected couples each of whom underwent two consecutive IVF/ICSI cycles, one with fresh and frozen and the next with frozenthawed epididymal spermatozoa. In this way, each couple served as its own control and the time between the IVF cycles was minimized to reduce the effect of age-related changes in maternal reproductive potential on the results. Similar to the majority of previous studies, we conclude that fresh and frozenthawed epididymal spermatozoa produce similar ICSI oocyte fertilization rates. Furthermore, in our observations of early embryo development, we noted no significant differences in early embryo quality with the two types of epididymal spermatozoa, unlike the observations made by Hutchon et al. (1998).
Despite the repeated measures design of this retrospective study, there are inherent limitations to our findings. Certain confounding variables may have influenced our results but the effects of these variables are difficult to analyse. The average increase in maternal age between the first and second IVF cycles was 1 year and therefore differences in maternal reproductive potential may exist. In addition, a comparison of pregnancy rates between the first and second IVF cycles may not accurately reflect differences in sperm quality because of an obvious, inherent selection bias: most couples proceeded to a second IVF cycle because of pregnancy failure in the initial cycle. Moreover, the higher pregnancy rate obtained with frozenthawed epididymal spermatozoa in the second cycle may simply directly reflect the application of knowledge about the female partner gained from the clinical experience of the first IVF cycle. This is exemplified by the fact that, on average, more oocytes were retrieved and more embryos were transferred in the second than the first IVF cycles. Despite these caveats, the similarities in oocyte fertilization rates and embryo quality between the two kinds of motile epididymal spermatozoa suggest that they are equal with respect to early ICSI outcomes.
In summary, this study provides strong evidence to support the notion that motile, cryopreserved and thawed epididymal spermatozoa are equal to freshly retrieved spermatozoa for ICSI in couples with obstructive azoospermia. It is hoped that these findings raise clinical awareness of the value and priority of sperm cryopreservation in all epididymal sperm aspiration procedures.
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Notes |
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References |
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Submitted on August 29, 2000; accepted on November 7, 2000.