MALE FACTOR. Liverpool Women s Hospital, Liverpool, England

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1 MALE FACTOR FERTILITY AND STERILITY VOL. 77, NO. 6, JUNE 2002 Copyright 2002 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Reproductive potential of fresh and cryopreserved epididymal and testicular in consecutive intracytoplasmic sperm injection cycles in the same patients Simon Wood, M.R.C.O.G., a Kevin Thomas, M.R.C.O.G., a,b Karen Schnauffer, B.Sc., a Stephen Troup, Ph.D., a Charles Kingsland, M.D., a and Iwan Lewis-Jones, M.D. a,b Liverpool Women s Hospital, Liverpool, England Received August 7, 2001; revised and accepted December 3, Reprint requests: Simon Wood, M.R.C.O.G., Reproductive Medicine Unit, Liverpool Women s Hospital, Crown Street, Liverpool L8 7SS, England (FAX: ; simon_j.wood@ virgin.net) a Reproductive Medicine Unit. b Department of Obstetrics and Gynaecology, University of Liverpool /02/$22.00 PII S (02) Objective: To determine if the cryopreservation of epididymal and testicular alters their reproductive potential by examination of patients who underwent consecutive cycles of ICSI using fresh and then cryopreserved. Design: Retrospective review. Setting: Tertiary care university hospital. Patient(s): One hundred sixty-two consecutive cycles of ICSI were analyzed. Thirteen patients were identified as having undergone treatment with freshly retrieved epididymal ; these patients subsequently underwent treatment with cryopreserved from that cycle. Eighteen patients underwent ICSI with freshly retrieved testicular ; these patients subsequently underwent treatment with cryopreserved from that cycle. Intervention(s): None. Main Outcome Measure(s): Fertilization rates and pregnancy rates. Result(s): The fertilizing capacity of epididymal remained unchanged after cryopreservation and subsequent thawing, with fertilization rates of 58% and 57% for fresh and cryopreserved, respectively. Testicular, however, showed a significant decrease in fertilizing capacity after cryopreservation when compared with freshly retrieved (52% and 71%, respectively). Pregnancy rates appeared unaffected by the cryopreservation of epididymal (fresh, 3/13; frozen, 2/13) or testicular (fresh, 2/18; frozen, 5/18). Conclusion(s): This study offers further evidence that motile epididymal retain their fertilizing capacity after cryopreservation. The data presented on testicular suggest that although cryopreservation may reduce the fertilizing capacity of testicular, there is no decrease in pregnancy rates. (Fertil Steril 2002;77: by American Society for Reproductive Medicine.) Key Words: Cryopreservation, epididymal, testicular,, ICSI The first pregnancy achieved by conventional IVF using aspirated from the epididymis of men with azoospermia was reported in 1985 (1), predating by 8 years the introduction by Van Steirteghem et al. of ICSI (2). Before the development of ICSI in 1993, fertilization rates for IVF with epididymal remained extremely low, and although fertilization was achieved in vitro using aspirated from the testes, no pregnancies were reported (3). After the introduction of ICSI, for which only one viable sperm is necessary for oocyte fertilization, the first successful pregnancy was achieved using extracted directly from the testis (4). Subsequently, the use of obtained directly from the testis has become a common procedure in assisted conception for patients with azoospermia (5 7). After these successes, many centers began to use cryopreserved surgically retrieved in addition to freshly retrieved sper- 1162

2 matozoa. Excess from this surgical retrieval or from other elective procedures, such as diagnostic biopsies to assess the presence or absence of or a planned pretreatment harvest of, were cryopreserved. The clinical benefits to both partners of cryopreservation are clearly evident: there is a reduction in the number of surgical retrievals (8, 9) and a reduction of the logistic complications of arranging concurrent and oocyte retrievals (9). The most crucial issue, however, is whether treatment with cryopreserved provides an acceptable alternative to treatment with fresh with regard to fertilizing capacity and pregnancy rates. There is little evidence to suggest that there is a difference in fertilization, cleavage, and implantation rates or pregnancy outcome between the use of cryopreserved and fresh retrieved from the epididymis (9 14), and there are advocates for using only cryopreserved epididymal (9). The efficacy of using cryopreserved extracted from the testes is slightly more questionable. Although the fertilization rates of cryopreserved extracted from the testes appear to remain acceptable, some studies have reported a small but significant reduction in both fertilization and implantation rates, with a corresponding reduction in live birth rate (15, 16), while other studies have shown no difference (17). Most of the above studies, however, used retrospective or historical data from unmatched control populations to compare outcomes. Four studies looked at patients undergoing treatment with previously cryopreserved epididymal from an earlier cycle of ICSI using fresh epididymal (13, 14, 18, 19). These studies all confirmed that previously cryopreserved epididymal showed no differences in fertilization rates, embryo cleavage rates, embryo quality, and pregnancy rates compared with that of fresh epididymal. However, none of these studies looked at the effect of cryopreservation and subsequent thawing on testicular. We wished to establish if trends seen previously in unmatched controls showed reductions in fertilization rates with cryopreserved testicular (15, 16). MATERIALS AND METHODS Patient Population During a 2-year period, 159 surgical retrievals were performed either using percutaneous epididymal aspiration (PESA) or, if unsuccessful, testicular extraction (TESE). Of these, 41 were performed electively and any harvested were cryopreserved. One hundred twenty-one retrievals were performed for obstructive azoospermia, and the retrieval rate for was 100%, with 33/121 (27%) being retrieved from the epididymis and the remainder from the testis. Thirty-nine TESE procedures were performed for nonobstructive azoospermia, as diagnosed by previous biopsy or raised FSH level 18 IU/L and testicular size 10 ml. Of these, 17 were successful in retrieving (43%). One hundred sixty-two cycles of ICSI were performed, of which 85 used fresh testicular, 33 used fresh epididymal, and 44 used frozen-thawed. Fifteen cycles used epididymal, and 29 used testicular. All patients who underwent an initial cycle of ICSI with freshly retrieved and a second ICSI cycle with cryopreserved retrieved during the first cycle were identified. Thirty-one patients met these criteria, of which 13 underwent two cycles with epididymal and 18 underwent two cycles with testicular. The data for each cycle with regard to oocytes collected, injected, and fertilized and pregnancy outcomes were analyzed by paired t-test and McNemar s test to assess differences between each of the two ICSI cycles. Spermatozoa Retrieval Methods PESA was performed using the method first described by Craft et al. in 1995 (20). After the epididymis was identified and secured between the thumb and index finger, the remaining fingers and palm were used to cup and stabilize the testicle. Epididymal fluid was aspirated blindly from the epididymis via the percutaneous puncture of the epididymis with a 19-gauge needle attached to a 20 ml syringe. The procedure was performed under general anesthesia, and the epididymal fluid aspirated was immediately examined at a magnification of 400 using Hoffman optics. If no motile were seen, the patient proceeded immediately to TESE. TESE was performed as an open biopsy, in which a small incision was made in the scrotal skin anterolaterally and the layers of tissue were opened through the spermatic fascia and the tunica vaginalis to the tunica albuginea, facilitating removal of seminiferous tubules. To extract from the tubules, the tissue was dissected and the individual sections of tubule milked with sterile needles (21). Oocyte Stimulation and Retrieval, ICSI, and ET Female patients underwent pituitary desensitization with a gonadotropin hormone releasing agonist (Suprefact; Hoechst, Frankfurt Am Mein, Germany) and ovarian stimulation with hmg (Menogon; Ferring GmBh, Kiel, Germany). Retrieved oocytes were processed in the laboratory in the manner described by Hillier et al. (22) and cultured in P1 medium (Irvine Scientific; Santa Ana, CA) supplemented with 10% v/v serum substitute supplement (SSS) (Irvine) under an atmosphere of 5% CO 2 in air at 37 C. After approximately 4 hours of incubation, the oocytes were mechanically denuded of their cumulus and coronal cells after brief exposure to hyaluronidase (Hyase; Vitrolife, Goteborg, Sweden). FERTILITY & STERILITY 1163

3 Epididymal were washed by centrifugation at 600 g in P1 medium for 10 minutes followed by resuspension in 1 ml of fresh medium. Testicular extracted from the seminiferous tubules were washed by centrifugation at 600 g in P1 medium for 10 minutes followed by resuspension in 3 ml of fresh medium. This sample was cultured overnight at 37 C in an atmosphere of 5% CO 2 in air and observed for motile the following morning. Only motile with normal morphology were used for ICSI, these being transferred to PVP (ICSI-100; Vitrolife) for immobilization before injection. Injected oocytes were cultured in P1 medium and examined for the presence of pronuclei approximately 18 hours after ICSI. Immediately before transfer of the embryos, all the embryos were assessed and graded according to standard criteria (22, 23). A maximum of three embryos were transferred into the uterus approximately 48 hours post-icsi. Successful implantation was determined by positive urinary -hcg and a clinical pregnancy by the presence of a fetal heartbeat on vaginal scan 4 6 weeks after ET. Cryopreservation and Thawing Excess epididymal and testicular that were not used for ICSI in the first cycle were cryopreserved (using SpermFreeze; FertiPro N.V, Beernem, Belgium) in 0.5 ml straws using a slow-freeze method (24). Straws were thawed rapidly at 37 C for 10 minutes. Cryoprotectant was removed with the dropwise addition of P1 medium followed by centrifugation at 600 g for 10 minutes and resuspension in 0.5 ml fresh medium. Statistical Analysis Statistical analysis was performed using the paired t-test to compare fresh and frozen outcomes for epididymal and testicular and McNemar s test to compare pregnancy rates. Independent t-tests and 2 tests were used to compare fertilization and pregnancy rates between epididymal and testicular. Differences were considered significant if P.05. RESULTS The mean age of the female patients treated with epididymal and testicular did not differ (31.8 and 30.9 years, respectively; P.568), nor did the average interval (and therefore duration of cryopreservation) between treatments (8.7 and 7.8 months; P.55) (see Table 1). There was no significant difference in number of oocytes collected or injected, number of grade 1 embryos per cycle, or number of embryos cryopreserved or transferred per cycle between any of the four groups. There was no significant difference (P.914) when comparing the fertilization rate for fresh and frozen epididymal (50/90 [58%] vs. 57/96 [57%] [P.914], respectively) (see Table 2). TABLE 1 Patients undergoing surgical sperm retrieval and ICSI. However, while there was no significant difference in fertilization rates of metaphase II oocytes injected between fresh epididymal and fresh testicular (50/90 [58%] vs. 91/126 [72%] [P.063], respectively), cryopreserved testicular showed a significant decrease in fertilization rates when compared with freshly retrieved (76/144 [53%] vs. 91/126 [72%] [P.009], respectively) (see Table 3). Clinical pregnancy rates with fresh epididymal were 2/13 compared with 3/13 with cryopreserved (P.9). Similarly, no difference (P.43) was noted between the clinical pregnancy rates with fresh (2/18) and cryopreserved (5/18) testicular. DISCUSSION PESA TESE No. of patients Average male age in years (range) 41.5 (29 54) 41.4 (28 61) Average female age in years (range) 31.9 (24 39) 30.9 (21 44) Interval between treatments in months (range) 8.69 (3 17) 7.77 (4 15) Note: Values are median with interquartile range. P values were not significant. PESA percutaneous epididymal aspiration; TESE testicular extraction. In the past 8 years, pregnancy rates with ICSI have risen dramatically, especially in those cycles that used surgically retrieved. However, pregnancy rates still remain at a level where most couples will require multiple TABLE 2 Patients undergoing treatment with fresh and cryopreserved epididymal. Fresh epididymal Frozenthawed epididymal No. of cycles No. of oocytes injected (mean 90 ( ) 96 ( ) SD) % Fertilization rate (range) 58 (33 100) 57 (0 90) % Cleavage rate % (range) 93 (50 100) 94 (33 100) No. of grade 1 embryos No. of embryos transferred per cycle Clinical pregnancy rate 2/13 3/13 Note: P values were not significant Wood et al. Matched cryopreserved ICSI cycles Vol. 77, No. 6, June 2002

4 TABLE 3 Patients undergoing treatment with fresh and cryopreserved testicular. Fresh testicular Frozen-thawed testicular No. of cycles No. of oocytes injected 144 (8 3.1) 126 (7 4.3) NS (mean SD) % Fertilization rate (range) 71 (50 100) 53 (25 100).009 a % Cleavage rate (range) 94 (75 100) 100 (NA).047 a No. of grade 1 embryos NS No. of embryos transferred NS per cycle Clinical pregnancy rate 2/18 5/18 NS a Statistically significant. NA not available; NS not significant. attempts at oocyte collection and ICSI to achieve pregnancy (12). For this reason, it was imperative to attempt to cryopreserve recovered for utilization in further ICSI cycles to reduce the need for further invasive surgical retrievals. Many studies have shown that the use of frozen thawed epididymal was not associated with a reduction in fertilization potential or pregnancy rates (18, 19). Four studies have sought to match couples undergoing consecutive treatments with fresh and frozen epididymal (13, 14, 18, 19). Although these studies used a mixture of methods for sperm collection, either PESA or the more invasive microscopic epididymal aspiration, the patients were matched for the major determinants of outcome by virtue of the comparative data coming from consecutive cycles. These studies have now established that epididymal retain normal fertilization and pregnancy potential after cryopreservation. In Hutcheon et al. s study of 17 ICSI cycles (18), the fertilization rates were identical, but the authors did note a reduction in the number of grade 1 embryos for transfer. However, the data presented above would support other workers (13, 14), suggesting that there is no decrease in embryo quality associated with the use of cryopreserved epididymal. The effect of cryopreservation on epididymal l parameters was initially reported by Bachtell et al. (25), who confirmed the suitability of cryopreserved for treatment. More recent studies have also demonstrated the effect of cryopreservation on epididymal, with reductions in concentrations of viable as well as a significant decrease in the percentage of motile from 20% to 10% (14). While the use of epididymal has been studied extensively, the clinical effect of using cryopreserved, P surgically retrieved testicular in patients matched for the major determinants of outcome has yet to be established. Although many studies exist to demonstrate that using frozen testicular is an acceptable treatment with no effect on fertilization rate (17), some studies have noted a small but significant drop in fertilization rates (15, 16). These studies were population based and not in matched patients, and it was considered appropriate in the present study to examine data from couples undergoing consecutive cycles to establish the true effect of cryopreservation on testicular. The present study confirms a significant decrease in the fertilizing capacity of cryopreserved testicular but no significant effect on embryo quality, the numbers of embryos available for transfer or cryopreservation, or pregnancy rates. This reduction in fertilization rate, therefore, clearly appears to have no impact on the clinical outcome of an ICSI. Although patients have been matched, it is clear that the cycles using cryopreserved occurred after previous treatment with fresh and thus the patients were older, which may affect pregnancy rates. The average interval after first treatment was around 8 months, with a range of intervals between 3 and 17 months. This difference is unlikely to significantly alter any of the variables measured. The inherent selection bias established between the fresh and cryopreserved cycles in terms of pregnancy rates could be said not to reflect true outcomes, as second cycles are more likely to occur in patients who have unsuccessful cycles using fresh. Further bias could be said to occur by applying clinical knowledge gained from the first cycle to optimize treatment in the second cycle in terms of ovarian stimulation regimens. However, comparison of the number of oocytes obtained and pregnancy rates in each of the four groups would suggest that no such bias is present. Although this study is not large, it may become increasingly difficult in the future to examine larger numbers of patients in individual units because of a lack of patients undergoing consecutive cycles of ICSI using fresh then cryopreserved. The consensus within the literature demonstrates no difference in fresh and cryopreserved epididymal and no clinical difference to the patient between fresh and cryopreserved testicular. The data presented above provide reassurance in support of the greater use of elective harvesting and cryopreservation of epididymal or testicular from the male. These data also support the harvesting and storage of at other procedures such as vasectomy reversal (26, 27) or diagnostic testicular biopsy. Such strategies serve to reduce the need for invasive surgery and the inconvenience to both patients and clinicians of trying to arrange concurrent oocyte and retrievals. FERTILITY & STERILITY 1165

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