Percutaneous epididymal sperm aspiration and intracytoplasmic sperm injection in the management of infertility due to obstructive azoospermia

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1 FERTILITY AND STERILITY Vol. 63, No.5, May 1995 Copyright It) 1995 American Society for Reproductive Medicine Printed on acid-free paper in U. S. A. Percutaneous epididymal sperm aspiration and intracytoplasmic sperm injection in the management of infertility due to obstructive azoospermia Ian Craft, F.RC.S., F.RC.O.G. Marinos Tsirigotis, M.D., M.RC.O.G. * Valerie Bennett, B.Sc. Mohamed Taranissi, M.RC.O.G. Yasser Khalifa, M.RC.O.G. Georg Hogewind, M.D. Natasha Nicholson, B.Sc. London Gynaecology and Fertility Centre, London, United Kingdom Objective: To evaluate the recovery rate of spermatozoa from the epididymis using a percutaneous aspiration technique and to examine the fertilization rate after intracytoplasmic sperm injection. Design: Prospective observational study. Setting: Private infertility clinic, London. Subjects: Twenty patients with obstructive azoospermia who each had an attempt at IVF. The sperm used for intracytoplasmic sperm injection was retrieved by percutaneous epididymal sperm aspiration in 16 patients. In one patient, microepididymal sperm aspiration was performed in addition because the quality of the sperm obtained by percutaneous epididymal sperm aspiration was not considered suitable for microinjection. In the remaining three patients, neither percutaneous epididymal sperm aspiration nor microepididymal sperm aspiration resulted in the recovery of sperm, which was obtained by testicular biopsy in one of them. Intervention: Assisted fertilization with intracytoplasmic sperm injection. Main Outcome Measures: Normal fertilization and pregnancy rates. Results: A total of 179 eggs were collected and 157 subsequently were microinjected. Normal fertilization occurred in 22 oocytes (14%) and the total number of embryos cleaved was 30. Twelve patients underwent ET in which three conceived (pregnancy rate 25% per transfer). The implantation rate was 10% and failed fertilization occurred in four cycles. Conclusion: Percutaneous epididymal sperm aspiration can be used successfully to recover sperm in men with obstructive azoospermia for use in assisted fertilization IVF cycles. The technique is simple, effective, and less traumatic compared with an open microsurgical operation. Fertil Steril 1995;63: Key Words: Obstructive azoospermia, percutaneous aspiration, assisted fertilization Male factor infertility has undergone significant changes in the last few years. The introduction of assisted fertilization (1-4) has revolutionized treatment to the extent that the old concept that sperm should go through the full length of the genital tract before it is able to achieve fertilization is no longer valid (5). Obstructive azoospermia has, to date, been treated with microsurgery, although IVF with epi- Received June 24, 1994; revised and accepted December 14, * Reprint requests: Marinos Tsirigotis, M.D., M.R.C.O.G., Cozens House, 112A Harley Street, London WIN laf, United Kingdom (FAX: ). didymal sperm for irreversible cases and those with congenital absence of the vas has been practiced (6). However, the results obtained from IVF using sperm aspirated from the epididymis have been poor until recently (7). Microepididymal sperm aspiration with scrotal exploration under general anesthetic has been used to retrieve sperm for use either with conventional IVF (8) or in assisted fertilization cycles with more encouraging results (9). Nevertheless, the technique involves a certain amount of trauma and postoperative morbidity, i.e., pain, hematoma formation, and infection may occur, and any subsequent surgery may be more complex because of postoperative adhesions and fibrosis. The aim of this study 1038 Craft et al. Percutaneous simplified sperm recovery in obstructive azoospermia Fertility and Sterility

2 Table 1 Indications for Microsurgical Sperm Retrieval and Origin of Sperm Used Sperm retrieved Percutaneous No. of epididymal sperm Microepididymal Testicular Etiology patients aspiration sperm aspiration sperm Sperm used Failed vasectomy reversal 12 10/12 Congenital absence of the vas 5. 4/5 Inflammatory obstruction 2 2/2:j: Unknown 1 III * Suitable sperm for intracytoplasmic sperm injection was not retrieved in two cases despite microepididymal sperm aspiration and testicular biopsy. t In this case, sperm was not retrieved by either percutaneous epididymal sperm aspirationimicroepididymal sperm aspiration and testicular sperm was used. 012* 0/2* PESA X 10 ollt lilt PESA X 4 TESTIS xl 1/1:j: PESA xl MESA X 1:j: PESA xl :j: Sperm was retrieved by both percutaneous epididymal sperm aspiration-microepididymal sperm aspiration but the microepididymal sperm aspiration sample was used. was to assess the recovery rate of spermatozoa from the epididymis and/or testes using a percutaneous aspiration technique, and the fertilization rate obtained with intracytoplasmic sperm injection. MATERIALS AND METHODS Between September 1993 and March 1994, 20 patients had percutaneous epididymal sperm aspiration to recover sperm for intracytoplasmic sperm injection. In 16 patients the sperm used was recovered by this technique; in the remaining 4 patients the sperm used was that obtained by microepididymal sperm aspiration or from testicular tissue. The indications for the sperm retrieval and the origin of the sperm used in the micromanipulation process are shown in Table 1. Patients were counseled about this novel way of recovering sperm and of its utilization by intracytoplasmic sperm injection and gave their informed consent. The mean age of the patients was 30.5 years (range 25 to 42 years). Protocols used for ovarian stimulation were the same as those used in conventional IVF and have been described previously (10). The ovarian response was monitored with vaginal ultrasonography and 10,000 IU hcg was administered when the leading follicles were > 20 mm in diameter. Transvaginal egg collection was performed under sedation 36 hours after hcg. The luteal phase was supplemented with natural 400 mg P pessaries 12 hourly (Cyclogest; Hoescht, Hounslow, United Kingdom). Sperm suitable for intracytoplasmic sperm injection was retrieved in 16 of 20 cases when percutaneous epididymal sperm aspiration was carried out. As is shown in Table 1, percutaneous epididymal sperm aspiration did not yield sperm in two cases and subsequent microepididymal sperm aspiration and testicular biopsy also failed to provide sperm suitable for microinjection. In a further case with congenital absence of the vas and epididymis, sperm was not retrieved by either the percutaneous epididymal sperm aspiration or microepididymal sperm aspiration procedure, and live motile sperm retrieved from the testes was used. (In one further case sperm was retrieved by percutaneous epididymal sperm aspiration but microepididymal sperm aspiration was undertaken to see if better quality sperm samples could be retrieved and the microepididymal sperm aspiration sample subsequently was used for microinjection). The FSH levels were normal in all men except two whose levels were elevated. Spermatozoa collected surgically were suspended in IVF culture medium (Medicult als, Copenhagen, Denmark). The surgical technique was undertaken using sterile precautions under either intravenous sedation or general anesthetic. The general anesthesia was favored in cases where scrotal exploration and microepididymal sperm aspiration or testicular biopsy were thought to be possibilities. Sperm aspiration was achieved by directing a 21- gauge butterfly needle (Venisystems; Abbott Ireland Ltd., Sligo, Republic of Ireland) connected to a syringe into either the head of the epididymis or, indeed, to the corpus, after immobilization of the testis by holding it stable beneath the thumb and index finger, which were placed immediately above it so that the epididymis was felt as a distinct structure overlying the proximal palmar aspect of the index digit. Suction was applied to the syringe, which were of various sizes (1 to 20 ml) and the needle was withdrawn gradually to a point where segments of fluid from the epididymis were seen entering the tubing of the microeffusion set attached to the butterfly needle. Steady, gentle, negative pressure was maintained until the segments of epididymal fluid Vol. 63, No.5, May 1995 Craft et ai. Percutaneous simplified sperm recovery in obstructive azoospermia 1039

3 Table 2 Sperm Parameters From Percutaneous Epididymal Sperm Aspiration Before and Mter Preparation Sperm (percutaneous epididymal sperm aspiration) Count (x10 6 /ml) Motility (%) Progression Abnormal forms (%) Retrieved 0.1 to 50 o to 30 0/4 to 2/4 50 to 95 Postpreparation Percoll Ca ++ -pentoxyfylline 0.5 to 30 o to 90 0/4 to 3/4 ceased to flow and an occlusive artery forcep was then applied across the microtubing before the needle was withdrawn from the skin. The aspirate then was washed out of the needle and tubing into a sterile Falcon tube (Becton Dickinson Ltd., Plymouth, United Kingdom) using IVF culture medium. This procedure can be performed as many times as necessary, and it is wise to do so on more than one occasion, because some tubules entered may have less favorable sperm than others. In some situations the fluid withdrawn is opalescent and milky, and it may be particularly so in those with an absent vas. It is infrequent that significant blood contamination occurs with this approach. The specimen is examined microscopically and the procedure is repeated again on the other side to maximize the amount and quality of sperm retrieved for microinjection. If repeated attempts to retrieve sperm by percutaneous epididymal sperm aspiration are unsuccessful, then scrotal exploration with microepididymal sperm aspiration and/or testicular biopsy may be carried out (Table 1). Sperm assessment was performed according to the recommendations by the World Health Organization (11) and prepared by a discontinuous Percoll gradient (12). The sperm was further enhanced by incubation with pentoxifylline (Sigma Chemical Company Ltd., Dorset, United Kingdom) for 30 minutes at a concentration of 2 mg/ml. It then was suspended in 2 ml of IVF culture medium and centrifuged for 5 minutes at 3,000 rpm. The resulting pellet was resuspended in 2 ml (3.2 /lmol) of calcium chloride 2-hydrate (Merck, Suffolk, United Kingdom) and again was recentrifuged as above. The sperm used was retrieved after assessment from the final pellet. The sperm parameters before and after preparation are shown in Table 2. Fertilization was determined by the presence of two pronuclei and the extrusion of the second polar body 16 to 18 hours after microinjection. Embryo transfer of up to three embryos was performed 48 hours after oocyte collection. Supernumerary embryos of good morphological quality, if available, were cryopreserved. A serum,b-hcg test was performed 12 days after ET. If positive, vaginal sonography was performed 3 weeks later to determine viability of the conception and the number of gestation sacs. RESULTS Of 179 oocytes, 157 (87.7%) were subject to intracytoplasmic sperm injection. Thirty-eight oocytes (24.2%) were damaged and, of the remaining 119 oocytes, 22 (18.5%) showed normal two pronucleus fertilization. Another 31 oocytes (26.0%) showed one pronucleus at the time of fertilization review and, of those, 9 cleaved to a two-cell stage and were included in the total of 30 cleaved embryos that were transferred subsequently. From the 16 percutaneous epididymal sperm aspiration-intracytoplasmic sperm injection cycles, 4 patients failed to achieve fertilization and the outcome of ET of the remaining 12 patients is shown in Table 3. The reason for failure to fertilize was thought to be predominantly due to poor oocyte quality rather than to extreme sperm parameters. Intrauterine pregnancy was confirmed by ultrasound in 3 of 12 patients who had ET and a singleton pregnancy was noted on each occasion. All pregnancies have been progressing satisfactorily. The implantation rate was 10% and no embryos were available for cryopreservation. There was no significant association between sperm parameters (concentration, motility, forward progression, or morphology) and fertilization or pregnancy rates in this series. In this small group of patients studied to date, no pregnancy occurred in a woman > 35 years of age, although we have other confirmed pregnancies using intracytoplasmic' sperm injection even in women > 40 years (9). DISCUSSION In the last decade, some severely asthenozoospermic couples have achieved pregnancies either using Table 3 Outcome of ETs in Percutaneous Epididymal Sperm Aspiration-Intracytoplasmic Sperm Injection Cycles Percutaneous Epididymal Sperm Aspiration-Intracytoplasmic Sperm Injection Per treatment cycle Per embryo transfer One embryo Two embryos Three embryos Implantation rate (sacs per embryo transferred) Embryos frozen No. of Pregnancies per Total* 3/16t (18.8) 3/12 (25) 1/3 (33) 0/2 217 (28.6) 3/30 (10) * Values in parentheses are percentages. t Sixteen percutaneous epididymal sperm aspiration-intracytoplasmic sperm injection cycles were calculated Craft et al. Percutaneous simplified sperm recovery in obstructive azoospermia Fertility and Sterility

4 conventional IVF or GIFT or zygote intrafallopian transfer (ZIFT) (13, 14). Theoretically, IVF or GIFT allows the limited number of available sperm a greater opportunity to achieve direct contact with the oocytes. In fact, the pregnancy rates observed after fertilization in such cases are not significantly different from that in couples with normal semen analyses (13, 15). The ability to achieve pregnancies by using these methods has led others to obtain sperm in men with azoospermia having a functioning testis from the site proximal to an irreparable obstruction and to use the sperm obtained in assisted conception cycles (16). Pregnancies resulting from the use of sperm obtained by microepididymal sperm aspiration has led to the conclusion that even sperm with severely reduced motility can fertilize oocytes if other obstacles are overcome. Good results were achieved by Silber and Asch (8) who reported a 21% pregnancy rate using sperm obtained by microepididymal sperm aspiration in 115 IVF -ZIFT cycles. The introduction of intracytoplasmic sperm injection has resulted in even better fertilization and pregnancy rates in cases with extreme male factor infertility (3, 17, 18). It therefore was inevitable and logical that sperm obtained by microsurgical methods from sites proximal to an obstruction also would be used for intracytoplasmic sperm injection in an attempt to achieve higher fertilization and pregnancy incidences. Factors that may influence the success ofivf after microepididymal sperm aspiration, e.g., the number of oocytes collected to increase the rate of fertilization, retrieval of enough sperm of good motility and free of contamination, and transfer of the embryos into the fallopian tube rather than into the uterus, may be less relevant when intracytoplasmic sperm injection is carried out because, in these cases, one is limited only by the number of living spermatozoa that can be microinjected in an attempt to achieve fertilization (9). Schlegel et al. (19) very recently reported a modified (open surgical) micropuncture technique using conventional in vitro insemination, partial zona dissection, or subzonal insemination (SUZI) and reported a 27.5% pregnancy rate per cycle with a similar monospermic fertilization rate (19% versus 18.5%). They indicated that the preliminary results from intracytoplasmic sperm injection appeared to be similar or better than those achieved with SUZI or partial zona dissection. However, our results cannot be compared with their study as the aspiration technique and micromanipulation process used were different. The cohort of patients also was smaller and included patients with elevated FSH levels possibly indicative of impaired spermatogenesis. Our experience of using percutaneous epididymal sperm aspiration as a means of obtaining sperm proximal to an irreparable obstruction with resultant pregnancies occurring after intracytoplasmic sperm injection suggests that formal open microepididymal sperm aspiration procedures are no longer required and, in the event of failing to obtain sperm by percutaneous epididymal sperm aspiration, attempts should be made to obtain testicular tissue by biopsy and isolation of sperm from the testis for microinjection purposes. We have evaluated different ways of obtaining testicular tissue, including open operation, wide-bore needle aspiration, drill biopsy, and, most recently, using a modified breast biopsy gun. It is our view that the latter approach will become the definitive one for obtaining testicular tissue if percutaneous epididymal sperm aspiration fails to obtain sperm because they are not present in the epididymal tubules. Indeed, one easily can make a routine practice of undertaking percutaneous epididymal sperm aspiration and percutaneous testicular biopsy on each occasion because the latter serves to provide evidence of the histologic status of the testis besides being a source of injectable sperm. There have been no postoperative complications in the series treated, and all patients have been discharged home within a few hours of the operation to resume their normal professional activities the subsequent day. The high patient acceptability and limited cost, together with the fact that it may be repeated on several occasions without the prospect of having associated fibrosis and other complications after open surgery, are advantages over microepididymal sperm aspiration. Percutaneous epididymal sperm aspiration also may be used as a diagnostic procedure before a planned assisted conception attempt and gives reassurance that sperm will be retrieved on the day of the intended IVF and intracytoplasmic sperm injection attempt. However, in those couples who elect to undergo percutaneous epididymal sperm aspirationtesticular biopsy to obtain sperm and in whom the situation is unclear before operation, a preoperative decision may have been made to use donor sperm after appropriate counseling if no sperm are retrieved from either location, i.e., epididymis or testis, and, in these circumstances, ET and/or cryopreservation can be planned. It is, as yet, not possible to assess definitively the efficacy of percutaneous epididymal sperm aspiration and intracytoplasmic sperm injection of achieving pregnancies from couples in whom the man has an irreparable obstruction until a larger series has been reviewed. However, when three embryos were transferred, two of seven patients became pregnant Vol. 63, No.5, May 1995 Craft et al. Percutaneous simplified sperm recovery in obstructive azoospermia 1041

5 (28.6%) with an implantation rate of 10%, which compares favorably with that reported in another series of 69 intracytoplasmic sperm injection cycles (18). However, the incidence of normal fertilization observed in this study (14%) was low when compared with that of a large series of IVF -intracytoplasmic sperm injection cycles reported by the same group (9, 18). It is of relevance that the number of oocytes that showed one pronucleus at fertilization review was 31 (20%). Of the 30 cleaved embryos that were transferred, 20 were the result of cleavage from 00- cytes that fertilized normally and the remaining 10 were from the one pronuclear group that subsequently showed normal cleavage. The decision to transfer embryos from the latter group was based on observations that up to 25% of these embryos have a normal chromosome constitution (20). These results indicate that percutaneous epididymal sperm aspiration can be used successfully to retrieve sperm in men who have irremediable obstructive azoospermia. The technique is simple, the equipment is minimal, and the training is shorter compared with open microsurgical approaches, which may lead to further fibrosis and difficulty in being repeated in view of the trauma involved. The operation is more cost effective, both in terms of surgical training and equipment required and expense to the patient, and the patients are able to return to work the following day without the risk of complications that may follow open surgical techniques. Acknowledgment. We thank Nahid Yazdani, M.D.U., and Michalis Pelekanos, B.Sc., for their contribution to this study. We are grateful to Life Force Research Ltd., London, United Kingdom and Organon, Cambridge, United Kingdom for their support with this study and to Miss Tracy Evans for preparing the manuscript. REFERENCES 1. 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