1 Centre for Reproductive Medicine, University Hospital, Dutch-speaking Brussels Free University (Vrije Universiteit Brussel), Laarbeeklaan 101, B-1090 Brussels, Belgium, 2 King Faisal Specialist Hospital and Research Centre, P.O. Box 3354, Riyadh 11211, Saudia Arabia and 3 St Luke's Hospital, 224 South Woods Mill Road, St Louis, MO 63017, USA
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Abstract |
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Key words: azoospermia/cryopreservation/epididymis/ICSI/MESA
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Introduction |
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Although the use of frozenthawed testicular spermatozoa for ICSI has been reported (Hovatta et al., 1995; Fischer et al., 1996
; Gil-Salom et al., 1996
; Podsiadly et al., 1996
; Romero et al., 1996
; Khalifeh et al., 1997
), sperm numbers retrieved after testicular biopsies tend to be limited as compared with those recovered after MESA, which makes cryopreservation far easier in the latter case. Pregnancies have been reported after ICSI with frozenthawed epididymal spermatozoa (Devroey et al., 1995
; Oates et al., 1996
; Holden et al., 1997
). However, results after ICSI using frozenthawed epididymal spermatozoa have never been compared to those using fresh epididymal spermatozoa and the question arises as to whether freezing epididymal spermatozoa may be considered generally useful.
In the present study we compared the results of ICSI with either fresh or frozenthawed epididymal spermatozoa in a large retrospective case series.
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Materials and methods |
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Microsurgical epididymal sperm aspiration (MESA)
A total of 176 MESA procedures were performed, 18 of which were carried out during a preliminary scrotal exploration with cryopreservation of the epididymal spermatozoa and 158 of which were carried out on the day of ovum retrieval for ICSI or the day before in a few patients. In the latter subgroup, 135 men had a single MESA procedure, 10 men had two procedures, five of which involved freezing of epididymal spermatozoa which turned out to be 100% immotile after thawing. In one couple the husband even underwent three MESA procedures.
The MESA procedure was performed under general anaesthesia. Using an operating microscope, the epididymis was carefully dissected and after haemostasis, using bipolar coagulation, an epididymal tubule was longitudinally opened by micro-scissors through a small opening in the serosa. In line with the findings of Silber et al. (1990), the proximal corporal or distal head region of the epididymis was first opened. The epididymal fluid was aspirated by means of a disposable tip from an intravenous cannula (Medicut, Sherwood Medical, Tullamore, Ireland) mounted on a 1 ml syringe (Plastipak; Becton Dickinson, Aalst, Belgium) filled with 0.1 ml HEPES-buffered Earle's medium supplemented with 0.4% human serum albumin (Belgian Red Cross, Brussels, Belgium). In a few MESA cycles, spermatozoa were aspirated by means of a mouth-controlled sterile hand-pulled glass pipette with a diameter of 200 µm. The aspirated epididymal fluid was then transferred into a Falcon test tube, filled with 0.9 ml of this Earle's medium. When motile spermatozoa, as assessed by peroperative microscopical examination of the aspirates, were recovered, no further epididymal incision was made and a maximum of fluid was aspirated in order to allow cryopreservation of as many epididymal spermatozoa as possible. The tubular incision was then closed by 90 nylon sutures. If microscopical assessment did not reveal any motile spermatozoa, a more proximal incision was made until motile spermatozoa were found. Sperm analysis was performed according to the World Health Organization (1992) guidelines taking dilution by Earle's medium into account, but for morphology assessment, the strict Tygerberg criteria were applied (Kruger et al., 1986). After analysis, the aspirated epididymal fluid was layered on a two-layer Percoll gradient (95 and 47.5%) in order not to rescue too many senescent spermatozoa. After washing, the spermatozoa were used for ICSI.
Cryopreservation of epididymal spermatozoa
The epididymal spermatozoa from 148 patients (91.3%) were cryopreserved. After Percoll preparation, one fraction of the epididymal aspirate was diluted to 2x106/ml spermatozoa in order to be able to freeze a maximum number of straws. The cryopreservation medium used contained glycerol at a concentration of 15%, egg yolk, citrate, glycine, glucose and antibiotics (Verheyen et al., 1993). After equilibration in a 37°C water bath for 10 min, the mixture was aspirated into 0.25 ml straws [Instruments de Médecine Véterinaire (IMV), l'Aigle, France]. The straws were then rapidly frozen in liquid nitrogen vapour by placing them horizontally 810 cm above the liquid nitrogen surface for 10 min (room temperature to 80°C; rate 10°C/min). The straws were then plunged directly into the liquid nitrogen (196°C). For thawing, the straws were removed from the liquid nitrogen and placed at 37°C for 10 min. In order to remove the cryoprotectant, thawed specimens were put on a two-layer Percoll gradient (9547.5%) and centrifuged for 20 min at 300 g. Afterwards, the 95% fraction was washed by centrifugation with Earle's medium at 1800 g for 5 min and the pellet was washed again and centrifuged in a 1.5 ml Eppendorf tube (1800 g, 5 min) in order to concentrate the spermatozoa. This step is necessary in order to collect most spermatozoa into a small microdroplet ready for injection.
Intracytoplasmic sperm injection (ICSI)
All female partners received ovarian stimulation treatment by a combination of gonadotrophin-releasing hormone agonist (GnRHa) and human menopausal gonadotrophins (HMG). When the patient had at least three follicles with a diameter of 17 mm and serum oestradiol concentrations of 1000 ng/l, administration of both GnRHa and HMG were discontinued and ovulation was induced with 10 000 IU human chorionic gonadotrophin (HCG).
All patients had a transvaginal ultrasound-guided ovum aspiration ~36 h after HCG injection under local anaesthesia. After recovery, oocytes were denuded from the surrounding granulosa cells and metaphase-II oocytes were microinjected as described previously (Van Steirteghem et al., 1995). Injected oocytes were cultured individually in 25 µl droplets of B2 medium (Bio-Mérieux, Brussels, Belgium) under oil. At 1618 h after injection, oocytes were examined under an inverted microscope for the presence of two polar bodies and two distinct pronuclei, as evidence of normal fertilization. After an additional 2430 h of in-vitro culture, embryos were examined under the microscope to assess their developmental stage and quality on the basis of their morphological aspects. Excellent embryos contained no anucleate fragments and showed equal or unequal blastomeres. Good embryos contained <20% anucleate fragments while fair embryos contained between 20 and 50% of anucleate fragments. A maximum of three cleaving embryos were then transferred into the uterine cavity, except in patients >40 years old where, on some occasions, more than three embryos were transferred. Supernumerary embryos were cryopreserved at the 48-cell stage using dimethylsulphoxide as a cryoprotectant (Van Steirteghem et al., 1994
). The luteal phase was supplemented by vaginal administration of 200 mg micronized progesterone three times daily (Utrogestan; Laboratoires Piette, Brussels, Belgium).
Pregnancy was diagnosed at least 10 days after transfer by rising HCG concentrations of 20 IU/ml in serum on two occasions. Preclinical abortion describes pregnancies where no gestational sac was detected and/or where hormone concentrations were falling. Clinical pregnancies were confirmed by the presence of a gestational sac containing a fetus with heart activity detected by transvaginal ultrasonography 46 weeks after transfer. Implantation rate was defined as the ratio of the number of gestational sacs containing a fetus with heart activity and the number of transferred embryos. Clinical pregnancies reaching 20 weeks of gestation were considered ongoing.
Statistical analysis
Outcome measures are expressed as means with 95% confidence intervals (CI). Whenever indicated, the 2-test was applied at the 5% level of significance. Statistical analysis was performed with the Medcalc statistical software package (Medcalc, Gent, Belgium),
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Results |
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No significant differences were observed in parameters of fresh or frozenthawed epididymal spermatozoa used for ICSI. After correction for dilution, the mean density was 9.8x106 per ml (95% CI 7.612.1) versus 10.8x106 per ml (95% CI 2.319.3) for fresh and frozenthawed spermatozoa respectively. Overall motility was respectively 15.1% (95% CI 12.118.1) versus 10.6% (95% CI 4.516.7). A mean of 13.0% of fresh spermatozoa had a normal morphology (95% CI 10.815.1) compared to a mean of 9.5% of frozenthawed spermatozoa (95% CI 4.115.0).
Table I shows the ICSI results. No significant differences in oocyte recovery were observed between the two groups. A total of 3649 oocytecumulus complexes (OCC) were aspirated, 279 of which showed a germinal vesicle and 134 of which were in metaphase I. A total of 3076 metaphase-II oocytes were injected, 292 of which were damaged (9.5%).
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As may be seen from Table II, a total of 245 transfers were performed. In the group with fresh epididymal spermatozoa the transfer rate was 92.3 versus 84.7% in the group with frozenthawed spermatozoa (P < 0.03 by
2-test). In the first group, 12 patients did not have embryos transferred because either no fertilization occurred (n = 6) or their fertilized oocytes did not develop any further (n = 6). In the second group, nine patients had fertilization failure while another nine patients had no cleaving embryos after normal fertilization (difference not significant). A total of 698 cleaving embryos were transferred. The overall pregnancy rate per ICSI cycle was significantly lower when frozenthawed epididymal spermatozoa were used. However, no significant differences were found either in clinical and ongoing pregnancy rates or in implantation rates. In the fresh epididymal sperm group 69 embryonic sacs were observed, including five triplet pregnancies and 13 twin pregnancies. In the group in which frozenthawed epididymal spermatozoa were used, one triplet and seven twin pregnancies were observed. In the group with fresh epididymal spermatozoa, 62 viable fetuses were observed at ultrasound (implantation rate of 14.8% per embryo transferred) and 42 pregnancies were ongoing (26.7% per ICSI cycle). The implantation rate was 12.2% in the group with frozenthawed epididymal spermatozoa (34 viable fetuses) and 25 pregnancies were ongoing (21.2% per ICSI cycle). These differences were not significant.
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Table IV shows the subset of patients who had ICSI both with fresh and frozenthawed epididymal spermatozoa. This comparison did not reveal any significant differences in fertilization rates, fertilization failure rate, cleavage rates or implantation rates.
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Discussion |
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In the present study we analysed retrospectively a larger consecutive case series of couples undergoing ICSI with epididymal spermatozoa, either fresh or frozenthawed, in order to assess whether the freezing and thawing of epididymal spermatozoa might jeopardize the outcome after ICSI.
Although the freezingthawing process may impair the quality and especially the motility of the epididymal spermatozoa, we observed no significant differences in quality between fresh and frozenthawed epididymal spermatozoa. This surprising finding results from the retrospective character of this study. Patients who had their epididymal spermatozoa thawed and in whom all spermatozoa were found immotile either had a repeat MESA (five such patients are included in this series) or a testicular sperm recovery on the day of the ovum retrieval.
Yet this happened only in patients who were scheduled for a second or third ICSI attempt with cryopreserved epididymal spermatozoa. This means that they had at least one ICSI cycle with frozenthawed epididymal spermatozoa. Another option for such patients with only immotile spermatozoa after thawing might have been to select immotile but vital spermatozoa by the hypo-osmotic swelling test as has been suggested for ejaculated spermatozoa (Casper et al., 1996; Tsai et al., 1997
; Verheyen et al., 1997
). However, this test may not be reliable because of freezing-thawing damage to the cell membrane of the epididymal spermatozoa (Esteves et al., 1996
).
A second prerequisite for adopting cryopreservation of epididymal spermatozoa as a valid alternative to repeat surgery or another sperm recovery technique, is that the outcome after ICSI with frozenthawed epididymal spermatozoa has to be comparable to that of ICSI with fresh epididymal spermatozoa. Although we found no differences in fertilization rates, we observed a higher proportion of ICSI cycles with complete fertilization failure after using frozenthawed spermatozoa than after using fresh epididymal spermatozoa. We also observed a slightly greater number of cycles with no embryonic development after the use of frozenthawed epididymal spermatozoa. The causes of these differences, which were not significant, are not clear.
Although the overall pregnancy rate (+ HCG) was lower when frozenthawed spermatozoa were used, the clinical pregnancy rate per ICSI cycle was comparable. This is the result of a higher proportion of biochemical pregnancies in the group with fresh epididymal spermatozoa. It may be that the freezingthawing process eliminates senescent spermatozoa that might otherwise be used as fresh spermatozoa for ICSI.
As for all pregnancies after ICSI, patients were asked to participate in the prospective follow-up study on ICSI (Bonduelle et al., 1996). In this small series of pregnancies after MESA-ICSI we observed no karyotype anomaly. In three babies born after ICSI with fresh epididymal spermatozoa, major congenital anomalies were observed. In the group in which frozenthawed spermatozoa were used, only two minor congenital anomalies were observed. These figures underline the need for further prospective follow-up of babies born after the use of epididymal spermatozoa.
In another smaller study, the general outcome after ICSI with frozenthawed spermatozoa was found comparable to that for ICSI with fresh epididymal spermatozoa (Oates et al., 1996). Others have compared ICSI with frozenthawed epididymal spermatozoa to ICSI with ejaculated spermatozoa retrospectively and they too observed no difference in outcome (Holden et al., 1997
).
Since the clinical pregnancy rates and implantation rates are comparable to those of ICSI with fresh epididymal spermatozoa, cryopreserving epididymal spermatozoa during any MESA procedure is advocated. This MESA procedure may be performed either during a scrotal exploration or at some other point by an andrological microsurgeon who has no direct access to ICSI. Since the outcome after microsurgical vasoepididymostomy is limited (Berardinucci et al., 1998), cryopreservation of epididymal spermatozoa for future ICSI may be performed whenever possible as a back-up whenever this procedure fails to restore fertility.
MESA may also be proposed as the primary sperm recovery technique in patients with obstructive azoospermia. Considering that other less invasive, less expensive and even simpler techniques are available to retrieve spermatozoa in such patients, e.g. PESA, we propose MESA as the first-line approach in those patients who may benefit from a scrotal exploration. Patients in whom the feasibility for reconstructive microsurgery has not been evaluated are a target group for MESA. Patients wishing to store a maximum of epididymal spermatozoa may be good MESA candidates too because at present no reliable data can be found in the literature concerning the possibilities of cryopreserving and using frozenthawed spermatozoa obtained after PESA. Although pregnancies have been reported after ICSI with frozenthawed testicular spermatozoa from patients with obstructive azoospermia, at present these data too are limited and patients may prefer MESA with cryopreservation as the method of choice by which to avoid repeat surgery.
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Acknowledgments |
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Notes |
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References |
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Submitted on June 1, 1998; accepted on September 23, 1998.