MALE FACTOR. Use of frozen-thawed testicular sperm for intracytoplasmic sperm injection

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1 FERTILITY AND STERILITY VOL. 73, NO. 3, MARCH 2000 Copyright 2000 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. MALE FACTOR Use of frozen-thawed testicular sperm for intracytoplasmic sperm injection Wolfgang Küpker, M.D., Ph.D.,* Peter N. Schlegel, M.D., Safaa Al-Hasani, V.M.D., Ph.D.,* Paolo Fornara, M.D., Ph.D., Rainer Johannisson, Ph.D., Jörg Sandmann, M.D., Thilo Schill, M.D.,* Monika Bals-Pratsch, M.D.,* Michael Ludwig, M.D.,* and Klaus Diedrich, M.D., Ph.D.* Medical University Lübeck, Lübeck, Germany; and Cornell Medical Center, New York, New York Objective: To determine the feasibility of using frozen-thawed testicular spermatozoa for intracytoplasmic sperm injection. Design: Prospective clinical study. Setting: A university hospital. Patient(s): One hundred seventy-five azoospermic men participating in a routine intracytoplasmic sperm injection program. Intervention(s): The men underwent testicular biopsy for cryopreservation of tissue to be used in consecutive intracytoplasmic sperm injection treatment cycles. Their female partners underwent controlled ovarian hyperstimulation for conventional IVF treatment. Main Outcome Measure(s): Fertilization and pregnancy rates. Result(s): In 77% of the patients, spermatozoa could be harvested from the testis by an open testicular biopsy technique and used for intracytoplasmic sperm injection after freezing and thawing of testicular tissue. Histopathologic evaluation revealed a Sertoli cell only pattern in 21%, maturation arrest in 60%, and hypospermatogenesis in 19% of the patients. In 2.9% of the patients, carcinoma in situ or a germ cell tumor was detected. In all patients, viable spermatozoa could be visualized after the tissue samples were thawed. One hundred thirty-five intracytoplasmic sperm injection treatment cycles were performed, with a fertilization rate of 45% and a clinical pregnancy rate of 30% per oocyte retrieved. Conclusion(s): The use of frozen-thawed testicular tissue allows ovarian stimulation of the female partner to be timed and avoids cancellation of ovum pick-up when spermatozoa cannot be retrieved. (Fertil Steril 2000; 73: by American Society for Reproductive Medicine.) Key Words: Azoospermia, TESE, ICSI, frozen-thawed testicular spermatozoa Received July 6, 1999; revised and accepted October 4, Reprint requests: Wolfgang Küpker, M.D., Ph.D., Department of Obstetrics and Gynecology, Medical University Lübeck, Ratzeburger Allee 160, Lübeck, Germany (FAX: ). * Department of Obstetrics and Gynecology, Medical University Lübeck. Department of Urology, Cornell Medical Center. Department of Urology, Medical University Lübeck. Institute of Pathology, Medical University Lübeck /00/$20.00 PII S (99) Intracytoplasmic injection of spermatozoa into the oocyte is an effective therapy for severe male subfertility that results in high fertilization and pregnancy rates. In patients with obstructive and nonobstructive azoospermia (i.e., congenital absence of the vas deferens, complete absence of the epididymis, severe forms of spermatogenic failure), fertilization and pregnancy can be accomplished using a combination of microsurgical epididymal sperm aspiration or testicular sperm extraction (TESE) and intracytoplasmic sperm injection (ICSI). In patients who do not have mature spermatozoa in their testicular tissue, immature spermatogenic cells, such as round and elongated spermatids, can be used successfully for ICSI (1, 2). Men with nonobstructive azoospermia often have small testes and an elevated FSH level. Clinically, these patients may be characterized as having testicular failure. Testicular biopsy reveals hypospermatogenesis, maturation arrest, or a Sertoli cell only pattern. Typically, small, focal areas of sperm production are present in these testes, which makes TESE possible with multiple biopsies. The use of frozen-thawed ejaculated spermatozoa is well established in insemination cycles and IVF programs. Epididymal gametes from microsurgical epididymal sperm aspiration procedures also have been frozen, thawed, and used successfully for ICSI (3). However, only a few case reports describe the use of 453

2 FIGURE 1 Open testicular biopsy (two biopsy sites). FIGURE 2 Extracted testicular tissue before freezing. frozen-thawed testicular spermatozoa obtained by TESE for ICSI. We report a series of 135 successful ICSI cycles in which we used frozen-thawed testicular spermatozoa. MATERIALS AND METHODS Male Patients The men in this study were characterized as having nonobstructive azoospermia based on the results of a clinical examination. Testicular biopsy was performed in 175 cases. An open testicular biopsy technique (Fig. 1) was used with general anesthesia. It began with scrotal exploration through a median raphe incision. To identify the anatomy, the entire testis and epididymis were delivered completely and examined with microscopic assistance using 8-fold to 15-fold magnification. Incision of the tunica albuginea was performed in an apparently avascular region to avoid testicular vascularization. Testicular parenchyma was harvested from different sites in the testis. Retrieval was performed routinely at two sites on each testis. The excised tissue was placed immediately into 5-mL Falcon tubes (Falcon 2003; Becton Dickinson, Franklin Lakes, NJ) containing 1 ml of Ham s F-10 medium. Institutional Review Board approval was obtained before the diagnostic and therapeutic procedures were started. All patients gave written consent for the study. The biopsy specimens (Fig. 2) were transported to an adjacent laboratory and minced in a petri dish (Falcon 3037) containing Ham s F-10 medium using sterile scissors to isolate individual seminiferous tubules. The fragmented tissue was assessed on microscope slides for the presence of free, motile spermatozoa under a phase-contrast microscope (Standard 20; Zeiss, Jena, Germany) at magnification. All the biopsy specimens were stored in Ham s F-10 medium for half an hour at 37 C before they were frozen. Before the tunica albuginea was closed using 4/0 polyglactin 910 (Vicryl; Ethicon, Hamburg, Germany) suture, 2 ml of heparinized saline solution was rinsed underneath it to minimize scarification. The scrotal incision was closed with 3/0 catgut sutures. Female Patients Ovarian hyperstimulation was performed with use of a GnRH agonist (Decapeptyl-Gyn-Depot; Ferring, Kiel, Germany or Zoladex-Gyn; Zeneca, Plankstadt, Germany) for pituitary suppression and hmg (Menogon; Ferring, Pergonal; Serono, Munich, Germany, or Humegon, Organon, Munich, Germany) in a long protocol (4). Ovulation was induced by the administration of 10,000 IU of hcg when the average size of the leading follicle was 20 mm. Oocyte retrieval was performed by vaginal ultrasound guided puncture of follicles 36 hours after hcg administration, under general anesthesia if required. Freezing of the Testicular Tissue For samples in which spermatozoa were identified in the wet preparation immediately after the TESE procedure, the 454 Küpker et al. Frozen-thawed testicular tissue in ICSI Vol. 73, No. 3, March 2000

3 biopsy specimens were frozen in up to 10 fractions for subsequent use in therapeutic ICSI cycles. The technique used was similar to that described by Salzbrunn et al. (5). Testicular tissue suspension samples were placed in 0.5 ml of HEPES-buffered medium (Sperm Freeze; Medicult, Hamburg, Germany) consisting of modified Earle s balanced salt solution with 0.4% human serum albumin and 15% glycerol as cryoprotectants. The samples were frozen immediately using a Planer Kryo10 III apparatus (Messer Griesheim, Griesheim, Germany). After 60 minutes, the samples were stored in liquid nitrogen. The cooling procedure was performed in liquid nitrogen vapor; the samples were cooled to 30 C within the first 5 minutes and then exponentially to 150 C over the next 55 minutes. Thawing of the Testicular Tissue After oocytes were obtained during a superovulated treatment cycle, testicular tissue samples known to contain spermatozoa were thawed and prepared for the ICSI procedure. The tissue was prepared using either an enzymatic (10 cases) or a mechanical (125 cases) approach. Enzymatic Method For enzymatic thawing, the frozen tissue suspension sample initially was placed in a small glass container containing warm water (37 C) for 3 5 minutes. The specimen then was transferred to 1 ml of Sperm Prep medium (Medicult) supplemented with 0.8 mg of collagenase, type AI (Sigma, Heidelberg, Germany) and 0.2 g of trypsin inhibitor (Sigma) and incubated for 2 hours in a controlled environment of 5% CO 2 at 37 C. All solutions were filtered through a 0.2- m Sartorius filter (Sartorius, Göttingen, Germany). The sample was digested for another 2 hours in the gas-controlled incubator. Then the sample was removed from the incubation solution and the tissue was dissected again. Larger particles were removed and the remaining tubuli were centrifuged in suspension at 800 g for 10 minutes. The supernatant was discarded and a small portion of the sediment was transferred into a droplet of medium to allow viable spermatozoa to swim out of the remaining tissue. No motility-enhancing substance was used. Spermatozoa subsequently were transferred into a 5- L droplet of Ham s F-10 medium. A single spermatozoon was immobilized and aspirated into the microinjection pipette from a 5- L droplet of 10% polyvinylpyrrolidone (Medicult) before being injected into an oocyte. Mechanical Method For mechanical thawing, tissue samples were placed in a 37 C water bath for 3 5 minutes. The samples then were washed two times in Ham s F-10 medium supplemented with 15% human umbilical cord serum. After being placed in a petri dish (Falcon 3037) containing Ham s F-10 medium, the tissue was minced using sterile scissors and a scalpel to gently tear apart the individual seminiferous tubules. The tissue then was incubated for another 3 5 hours in Ham s F-10 medium. The supernatant was transferred into 2-mL Eppendorf tubes and centrifuged at 500 g for 1 minute. The pellet was resuspended in 3 L of Ham s F-10 medium. One milliliter of this suspension was transferred into a petri dish (Falcon 3037) containing one droplet of Ham s F-10 medium and one 5- L droplet of polyvinylpyrrolidone surrounded by four droplets of Ham s F-10 medium for the oocytes under mineral oil. Preparation of the Oocytes The oocytes were treated with 0.5% hyaluronidase (Sigma) for 10 seconds for enzymatic removal of the cumulus oophorus cells. Under stereomicroscopic guidance at 50 magnification, cells of the corona radiata were removed mechanically with the aid of a 200- m Pasteur pipette. Then the maturity of the oocytes was determined. Only oocytes in metaphase II were used for the ICSI procedure. The ICSI Procedure The preparation of the holding and injection pipettes, and the injection procedure itself, were described previously (6, 7). In brief, the injection pipette was filled with a small amount of polyvinylpyrrolidone and a free, motile spermatozoon was aspirated head-first from 5 L of Ham s F-10 culture medium into the pipette. The single sperm then was placed, without any tissue debris, in another droplet of polyvinylpyrrolidone and immediately immobilized. The sperm was aspirated again into the injection pipette tail-first and injected into an oocyte. Sixteen to 18 hours after injection, the oocytes were examined for the presence of pronuclei and polar bodies. If 3 oocytes showed normal fertilization, these preembryos were cryopreserved. The embryos were transferred into the uterine cavity after an additional 24 hours. Luteal support was provided with either hcg or natural micronized progesterone (600 mg/d). Histopathologic Evaluation One tissue sample from each testis was immersed in 5.5% glutaraldehyde for 2 hours and postfixed in osmium tetroxide for an additional 2 hours. Then it was dehydrated in ethanol and propylene oxide before being embedded in a polymerization mixture of Epon (Epon 812; Hert, Munich, Germany). Semithin sections were cut on an ultramicrotome with diamond knives and stained with toluidine blue/pyronin according to the method of Johannisson et al. (unpublished data). RESULTS A total of 175 azoospermic patients were evaluated and found to have nonobstructive azoospermia. None of these 175 patients had karyotypic abnormalities. In 77.1% (135/ 175) of the patients, spermatozoa were identified in a wet preparation after TESE, whereas in 22.9% (40/175), no FERTILITY & STERILITY 455

4 TABLE 1 Results of ICSI using frozen-thawed testicular spermatozoa. Variable Value No. of cycles 135 Age range (y) No. of oocytes 1,658 No. of injected oocytes 1,335 No. of fertilized oocytes (%) 605 (45.3) No. of ETs (%) 129 (96.0) No. of pregnancies (%)* 40 (29.6) Note: Rate per cycle. spermatozoa could be found. In 135 patients, spermatozoa were detected and used for ICSI in a programmed ovarian stimulation cycle for the female partner. The histologic findings included a Sertoli cell only pattern in 21%, maturation arrest in 60%, and hypospermatogenesis in 19% of the patients. In 5 (2.9%) of the 175 patients, histopathologic evaluation revealed a neoplasm: carcinoma in situ in 3 patients, a seminoma in 1 patient, and a Leydig cell tumor in 1 patient. Unilateral orchiectomy was performed immediately for the patient with a seminoma, and testicular tissue was obtained from both testes and frozen. Only one postoperative complication, an acute scrotal hematoma, was noted. One hundred thirty-five ICSI cycles were attempted using frozen-thawed spermatozoa. After thawing the testicular tissue and incubating it for approximately 5 hours, motile spermatozoa were found in all cases. After the injection of 1,335 metaphase II oocytes, a fertilization rate of 45% per injected oocyte was calculated (Table 1). In these 135 treatment cycles, 129 transfers were performed and 40 viable clinical pregnancies resulted that were confirmed on ultrasound examination. Twenty-nine women were delivered of healthy infants (5 twins and 24 singletons). Karyotyping of the products of conception after 11 spontaneous abortions showed no chromosomal aberrations (Table 2). TABLE 2 Pregnancy outcome after ICSI with frozen-thawed testicular spermatozoa. Variable Value No. of patients who conceived 40 No. of miscarriages 11 No. of singletons (delivered) 24* No. of twins (delivered) 5* Note: No malformations or chromosomal abnormalities. * Out of these 40 patients, all had delivered except the 11 miscarriages. DISCUSSION It was hypothesized as early as 1913 that a testicular biopsy might yield viable spermatozoa for an extracorporal treatment (8). Jow et al. (9) recently reported that spermatozoa could be found in a single testicular biopsy sample in 34% of men with nonobstructive azoospermia. Schoysman et al. (10, 11) and Devroey et al. (12, 13) reported fertilization and pregnancies after ICSI using testicular spermatozoa. Testicular sperm extraction can be used to retrieve spermatozoa from men with nonobstructive azoospermia in up to 70% of attempts. The use of a combination of TESE and ICSI with fresh testicular spermatozoa from azoospermic men has been reported at multiple centers (3, 14 17). The overall fertilization rate (43% 47%) has been slightly lower than that obtained with the use of fresh ejaculated spermatozoa. Clinical pregnancies were achieved in 20% 50% of sperm retrieval attempts. However, only anecdotal cases involving the use of frozen-thawed testicular spermatozoa have been reported (18 21). Pregnancies were achieved in these cases, and the average fertilization rate was 52%, which is comparable to the fertilization rates that are obtained when spermatozoa from fresh testicular biopsy specimens are used. In this study, 135 ICSI treatment cycles were performed using frozen-thawed testicular spermatozoa according to the protocols described. A total of 1,335 metaphase II oocytes were injected, resulting in a fertilization rate of 45%. The clinical pregnancy rate of 30% is comparable to the pregnancy rate obtained at our institution when ICSI is performed with fresh ejaculated spermatozoa (28%). One advantage of using frozen-thawed testicular tissue to obtain spermatozoa for ICSI is that the pattern of spermatogenesis can be evaluated before ovarian stimulation of the female partner is undertaken. No further operative intervention on the male partner is needed if the couple has to undergo subsequent treatment cycles to achieve a pregnancy. This means that for many couples, when no spermatozoa can be obtained from the testis, unnecessary ovarian hyperstimulation can be avoided. However, in some patients with extremely limited spermatogenesis, biopsies of multiple sites within the testis may be needed to obtain spermatozoa or spermatids, and this will have adverse effects on the integrity of the residual testicular tissue. Schlegel and Su (22) demonstrated both transient and permanent adverse effects of the TESE procedure. In a series of 64 men who underwent TESE, all of them had evidence of inflammatory changes or hematoma 3 months after the procedure. Moreover, impaired testicular blood flow was detected by Doppler sonography, with complete devascularization in patients who underwent multiple biopsies. When a second TESE procedure was performed within 6 months of an initial successful procedure, spermatozoa could be harvested in only 25% of cases. 456 Küpker et al. Frozen-thawed testicular tissue in ICSI Vol. 73, No. 3, March 2000

5 FIGURE 3 Sertoli cell only pattern with focal spermatogenesis. Toluidine blue/pyronine stain. Original magnification, 1,000). FIGURE 4 Protocol for freezing and thawing of testicular tissue. These physiologic consequences for the testis and spermatogenesis must be taken into account when considering repeated TESE procedures. We perform only two biopsies on each testis in an avascular region; we have found this to be adequate to determine the status of spermatogenesis and to cause as little trauma as possible. Despite the presence of a Sertoli cell only pattern on histologic evaluation, small areas of spermatogenesis were detected in many of our patients (Fig. 3). Bilateral testicular biopsy is preferred to optimize sperm retrieval and diagnostic evaluation of the testis. It has been reported that 2% 3% of men with severe male subfertility have testicular carcinoma in situ (23, 24). In addition, small seminomas occasionally are detected in subfertile patients (25). Of the 175 patients in this study, 2.9% had a concomitant testicular neoplasm. Therefore, precise histologic evaluation by a pathologist is strongly recommended before any further treatment is undertaken. In addition, a careful andrologic evaluation with a rigorous physical examination and/or ultrasonographic examination of the testis is needed before treatment. In this study, a planned treatment strategy of testicular sperm cryopreservation was used (Fig. 4). Our results indicate that the clinical pregnancy rate is similar when frozen-thawed testicular spermatozoa or fresh gametes are used for ICSI. The fertilization rate was slightly lower compared with that obtained with freshly ejaculated spermatozoa, but this may reflect the source and immaturity of the spermatozoa rather than the freezing procedure, and it did not affect the clinical pregnancy rate. We saw no evidence that testicular tissue or spermatozoa were damaged by the process of freezing and thawing. In all cases, sperm motility of some degree was present after thawing and subsequent incubation in medium. Cryopreservation of ejaculated spermatozoa has been suspected to result in an increased incidence of broken sperm necks after thawing, resulting in a lower fertilization capacity, possibly resulting from dysfunction of the essential paternal centrosome that is located in the neck region (26). However, further studies that include electron microscopy are needed to clarify this question. The clinical outcome of normal, undisturbed pregnancies supports the feasibility of a protocol of planned biopsy with tissue cryopreservation. In all 135 patients in whom spermatozoa were identified in the testicular wet preparation, viable (i.e., motile) frozen-thawed sperm subsequently were available for ICSI. In cases where no sperm could be obtained, an unnecessary ovarian hyperstimulation cycle was avoided. One could question whether additional, more extensive biopsies might have identified some spermatozoa in the cases in which no sperm were found. If this were true, these patients might have been denied an opportunity to conceive a pregnancy because of our use of a limited combined diagnostic and therapeutic biopsy. However, our TESE retrieval rate is comparable to that reported in other series of men with nonobstructive azoospermia by Devroey et al. (14), Kahraman et al. (15), and Schlegel et al. (17). In summary, the use of frozen-thawed testicular spermatozoa for ICSI yields acceptable fertilization and pregnancy rates. Freezing the testicular tissue guarantees paternal gametes for subsequent ovarian stimulation cycles and the need for only one combined diagnostic and therapeutic testicular biopsy procedure. A clinical pregnancy rate of 30% was obtained per ICSI attempt using frozen-thawed testicular spermatozoa extracted from men with nonobstructive azoospermia. FERTILITY & STERILITY 457

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