Intentional cryopreservation of epididymal spermatozoa from percutaneous aspiration for dissociated intracytoplasmic sperm injection cycles

Intentional cryopreservation of epididymal spermatozoa from percutaneous aspiration for dissociated intracytoplasmic sperm injection cycles1, KOK-MIN SEOW 1,2, SHIH-CHIA HUANG
1, MEI-LING HSIEH 1 AND JIANN-LOUNG HWANG
1,3
From the 1Department of Obstetrics and Gynecology, Shin Kong Wu Ho-Su Memorial Hospital, 2College of Medicine, Fu Jen Catholic University and the 3Department of Obstetrics and Gynecology, Taipei Medical University, Taipei, Taiwan
Acta Obstet Gynecol Scand 2004; 83: 745–750. # Acta Obstet Gynecol Scand 83 2004
Background. To investigate the possibility of cryopreservation of spermatozoa obtained from percutaneous epididymal sperm aspiration (PESA) in patients with obstructive azoospermia and the feasibility of intentional dissociation of PESA and intracytoplasmic sperm injection (ICSI) cycles. Methods. Fifty-six patients with obstructive azoospermia underwent diagnostic PESA before ovarian stimulation. If spermatozoa were found, they were frozen for subsequent ICSI. The outcome was compared with 17 fresh PESA/ICSI cycles. Results. Among the 56 patients, diagnostic PESA obtained spermatozoa in 51 patients. The mean motility of the spermatozoa decreased from 15.2% to 4.2% after freezing and thawing. These patients underwent 96 frozen PESA/ICSI cycles. The rates of fertilization, implantation and clinical pregnancy for frozen–thawed spermatozoa (71.6, 14.0 and 40.6%, respectively) were similar to those for fresh spermatozoa (69.2, 13.2 and 41.2%, respectively). Conclusions. Sufficient numbers of spermatozoa can be obtained for cryopreservation through PESA and the spermatozoa work well after thawing. The strategy of performing diagnostic PESA before ovarian stimulation and freezing the recovered spermatozoa for subsequent ICSI is feasible for patients with obstructive azoospermia.
Key words: azoospermia; cryopreservation; ICSI; MESA; PESA
Submitted 17 February, 2003 Accepted 3 August, 2003
Azoospermia is found in 10% of male infertility and obstruction of the male genital tract is responsible for 20% of azoospermia (1). For patients with obstructive azoospermia, microsurgical repair can
result in spontaneous pregnancies in some of them. In the others in whom the microsurgery fails, assisted reproductive technologies are needed. The technique of intracytoplasmic sperm injection (ICSI) using sur- gically retrieved sperm from the epididymis or testis has revolutionized the treatment for obstructive azoospermia. Various sperm retrieval techniques, including microsurgical epididymal sperm aspiration (MESA), percutaneous epididymal sperm aspiration (PESA), testicular sperm aspiration (TESA) and testicular biopsy, have been reported to obtain spermatozoa for the ICSI procedure.
Abbreviations: ICSI: intracytoplasmic sperm injection; MESA: microsurgical epididymal sperm aspiration; PESA: percutaneous epididymal sperm aspiration; TESA: testicular sperm aspiration; FSH: follicle stimulating hormone; LH: luteinizing hormone; CBAVD: congenital bilateral absence of vas deferens; HTF: human tubal fluid; HOS: hypoosmotic swelling.
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MESA was the first surgical sperm retrieval method involving the epididymis (2,3). However, this procedure requires expensive microsurgical equipment and specially trained urologists and is associated with many complications such as bleeding, hematoma, postoperative pain, infec- tion and fibrosis. As an alternative to MESA, PESA was introduced with the characteristics of minimal invasiveness, shorter operation time and simplicity (4–6). Several studies have shown PESA to be a safe and effective method of sperm retrieval, with a successful ICSI cycle rate in the range of 80–100% (4–6).
Usually, sperm and oocyte retrieval are per- formed on the same day. However, the various sperm retrieval techniques may not always be successful and many couples require more than one ICSI cycle. In many reproductive centers, especially those in the developing countries, experienced urologists capable of performing the more invasive sperm retrieval procedures may not be available at all times. If no spermatozoa can be recovered and no donor sperm are available, it is obvious that the cycle has to be canceled. It there- fore seems logical and financially prudent to per- form sperm retrieval in advance and cryopreserve sperm for ICSI at a later date.
Some authors have advocated that MESA and sperm cryopreservation should be performed separately and before oocyte retrieval (7,8). This approach assures the availability of sperm for ICSI and avoids the need for repeated sperm retrieval. Several studies have shown that the use of frozen–thawed epididymal spermatozoa obtained by MESA produced fertilization and preg- nancy rates comparable to those obtained using fresh ones (8–12).
Reports on the use of frozen–thawed epididy- mal spermatozoa obtained by PESA are scarce, and there has been concern about the efficiency of cryopreservation of PESA-derived spermatozoa (10,13). Hutchon et al. first reported that there were no significant differences between the fertil- ization, implantation and pregnancy rates with fresh or cryopreserved sperm retrieved by PESA (14). Three similar studies have been reported (11,15,16). However, the case numbers were small, and it is not known how many ICSI cycles the frozen PESA-sperm can provide. Since September 1996 we have initiated a strategy of performing diagnostic PESA for obstructive azoospermia before ovarian stimulation, and freezing the motile spermatozoa obtained for subsequent ICSI cycles. The aim of this study was to investigate the feasibility of this strategy and the effectiveness of cryopreservation of sperm- atozoa obtained from PESA.
Materials and methods
Patients
This was a prospective consecutive case series of infertile couples with obstructive azoospermia. The combination of sperm retrieval and ICSI was offered to these couples. The clinical work- up of the male partners with azoospermia included history taking, a thorough physical examination, repeated (at least three) semen analyses, and serum follicle stimulating hormone (FSH), luteinizing hormone (LH) and testosterone measurements. Serum prolactin and transrectal ultrasonography were performed when indicated. Patients with elevated FSH levels, atrophic testis, abnormal karyotyping or biopsy-proven nonobstructive azoospermia were excluded from this study. The female partners received basic work-up including hysterosalpingography, serum FSH, LH and pro- lactin, as well as laparoscopy if indicated.
From October 1995 to August 1996, before we initiated the policy of electively cryopreserving PESA-derived sperm, we performed PESA on the day of oocyte retrieval. There were 17 patients with obstructive azoospermia who had successful epididymal sperm retrieval (fresh PESA group).
From September 1996 to June 2002 we per- formed diagnostic PESA on the male partners with obstructive azoospermia before ovarian stimulation. If motile spermatozoa were found, they were cryopreserved for subsequent ICSI cycles. If PESA failed to retrieve spermatozoa, the patients were offered TESA or testicular biopsy. There were 56 patients in this group (fro- zen PESA group). The etiology for the obstruc- tion included: congenital bilateral absence of vas deferens (CBAVD, n¼ 28), failure or refusal of vasectomy reversal (n¼ 13), epididymal or vas obstruction (n¼ 11), and idiopathic (n¼ 4).
PESA and sperm freezing and thawing
The PESA procedure was modified from that described by Craft et al. (6). In brief, while hold- ing the testicle between the thumb and the index finger, the epididymis was palpated and a 21-gauge butterfly needle was passed into the epididymis. The butterfly needle was attached to a 20-mL syringe containing 2 mL of HEPES- buffered human tubal fluid (HTF) medium (Irvine Scientific, Santa Ana, California, USA). A negative pressure in the system was created by pulling the plunger. The tip of the needle was moved gently back and forth within the epididy- mis until columns of slightly opalescent fluid were seen coming into the tubing. As much epididymal
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fluid as possible was aspirated; and before remov- ing the needle from the epididymis, a small artery forceps was used to clamp the tubing. When no more aspirate could be obtained, the needle was removed from the epididymis. The aspirate was examined for the presence of motile spermatozoa under an inverted microscope (Diaphot 300; Nikon Corp., Tokyo, Japan). Motility was scored as follows: a¼ rapid forward progressive moti- lity; b¼ slow progressive motility; c¼ nonpro- gressive, twitching spermatozoa; d¼ immotile spermatozoa. PESA was repeated on the same side and/or the other side until a satisfactory amount of motile spermatozoa were deemed to have been recovered.
The aspirates were pooled together and were centrifuged at 1800g for 5 min The resulting pellet was resuspended with an adequate amount of HTF medium and prepared for freezing. The final concentration of spermatozoa was adjusted to 2–3 motile spermatozoa per high power field (400). Each cryotube (Nunc, Roskidle, Denmark) contained 0.5 mL of specimen, and an equal volume of Test Yolk Buffer freezing medium (Irvine Scientific) was added dropwise. The cryotubes were held at room temperature for 10 min, then at 4 C for 10 min, followed by place- ment in liquid nitrogen vapor for another 10 min. Finally, they were immersed in the liquid nitrogen at 196 C for storage. For thawing, one or more of the cryotubes were removed from the liquid nitrogen and held at room temperature. The thawed specimen was mixed with 4 mL of HTF medium and centrifuged at 1800g for 5 min. The resulting pellet was washed and centrifuged two more times. Finally, the resulting pellet was resuspended in 0.3 mL of HTF medium and used for ICSI.
Ovarian hyperstimulation and oocyte retrieval
Afterpituitarydesensitizationwithbuserelinacetate (Supremon; Hoechst, Frankfurt, Germany), the female partners were stimulated with human menopausal gonadotropin (Humegon; Organon, Oss, the Netherlands) or FSH (Metrodin; Serono, Rome, Italy). Human chorionic gonadotropin (Pregny1; Organon, Oss, the Netherlands), 10 000 IU, was administered when at least two follicles had reached the size of 18 mm with an appropriate serum estradiol level. Transvaginal ultrasound-guided oocyte retrieval was per- formed 34–36 h later.
Intracytoplasmic sperm injection
Two 10-mL wash droplets were placed at the per- iphery of the ICSI dish (Falcon 1006, Becton
Dickinson, Lincoln Park, New Jersey, USA). The wash drops contained HEPES-buffered HTF medium with 0.5% human serum albumin (Irvine Scientific). One microliter of the sperm suspension was dropped carefully into one wash droplet. A few spermatozoa were picked up with an injection pipette and moved into another wash droplet to decrease the contamination of cellular debris. Only motile spermatozoa with normal morphology were used for ICSI. The ICSI proce- dure was as described by Palermo et al. (17).
Assessment of fertilization, embryo development and embryo transfer
Injected oocytes were cultured in P1 medium (Irvine Scientific) supplemented with 10% syn- thetic serum substitute (SSS) (Irvine Scientific) in an atmosphere of 5% CO2 in air and were examined for the formation of two pronuclei about 18 h after ICSI. Embryo transfer was per- formed on day 2 or 3. All patients received luteal support with 600 mg micronized progesterone (Utrogestan; Laboratoires Piette International S.A., Brussels, Belgium) daily per vagina starting from the day after oocyte retrieval. Clinical preg- nancy was defined as a visible fetal heart beat on ultrasonography at 7 weeks of gestation.
Statistical analysis
Because of a large discrepancy in cycle numbers between the two groups, the Mann–Whitney U-test, applied two-tailed at the 5% level of significance, was used for the comparison of means. Values are expressed as mean standard deviation (SD). Comparison of percentages was performed with the 2-test. p< 0.05 was consi- dered as statistically significant. Analyses were performed using the SPSS statistical package (SPSS Inc., Chicago, IL, USA).
Results
The mean ages of the male partners were 37.6 8.1 years in the frozen PESA group and 38.4 10.3 years in the fresh PESA group, and the mean ages of the female partners were 32.3 4.6 and 32.1 5.3 years, respectively.
Of the 56 attempts of PESA in the frozen PESA group, we failed to retrieve spermatozoa in five patients (8.9%). After scrotal exploration, they were found to have severely damaged epi- didymis (n¼ 1), rete testis obstruction (n¼ 1) and nonobstructive azoospermia (n¼ 3). Testicular biopsy was performed in these five patients and
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was able to find spermatozoa for ICSI. They were not included in this study. In the 51 successful PESA attempts, the mean number of cryotubes stored per patient was 6.5 (range 3–12). No major complication occurred except for scrotal ecchym- osis in one patient, which was relieved by ice packing.
Characteristics of spermatozoa before and after freezing
The mean motilities of fresh spermatozoa were 16.3% (range 3–50%) and 15.2% (range 2–51%) in the fresh and frozen PESA groups, respectively. As the final volumes of epididymal aspirates after dilution with HTF medium were usually very low, it was difficult to estimate the actual concen- trations of spermatozoa. After freezing and thaw- ing, the mean motility of the spermatozoa decreased dramatically to 4.2% (range 0–20%), with most of the surviving spermatozoa being grades b and c, and rarely grade a. Only motile spermatozoa with normal morphology were selected for ICSI. In two patients no motile sper- matozoa could be found from all the stored cryo- tubes after thawing (2/51¼ 3.9%). The sperm motilities of these two patients before cryopreser- vation were 10% and 25%, respectively. After performing the hypoosmotic swelling (HOS) test, there were still no viable spermatozoa. Sal- vage PESA was performed immediately, and enough motile spermatozoa were obtained for ICSI. These two patients were excluded from the study. In the other 49 patients, one cryotube was sufficient from each patient after thawing for each ICSI attempt. These patients underwent 96 ICSI cycles during the study period.
ICSI results and pregnancy outcomes
The 49 couples in the frozen PESA group under- went 96 ICSI cycles. In the fresh PESA group, 17 ICSI cycles were performed in 17 couples. Table I
shows the results of ICSI for the two groups. The mean numbers of oocytes retrieved, injected oocytes and embryos transferred were all compar- able between the two groups. The fertilization rate, implantation rate and clinical pregnancy rate using frozen–thawed epididymal spermatozoa were similar to those achieved following the use of fresh epididymal spermatozoa.
Table II reveals the distribution of the 96 ICSI cycles in each ICSI attempt in the frozen PESA group. The average number of ICSI cycles was 1.96 per patient, with a maximum of four cycles in one patient. The pregnancy rates in the first three ICSI attempts were similar. This implies that enough spermatozoa can be obtained for up to three ICSI cycles.
In the 39 pregnancies in the frozen PESA group, there were 29 term deliveries (26 single- tons and three sets of twins) and four preterm deliveries (all twins). In the fresh PESA group, 17 ICSI cycles resulted in seven pregnancies. There were four term pregnancies (all singletons) and two preterm deliveries (both twins). Altogether, 48 babies have been born so far (40 in the frozen PESA group and eight in the fresh PESA group), and only one has gross anomaly (postaxial polydactyly).
Discussion
To date, this is the largest series to evaluate the outcome of ICSI using frozen–thawed spermato- zoa retrieved by PESA. This study shows that PESA can obtain enough spermatozoa for cryo- preservation, and the outcome with the frozen– thawed epididymal spermatozoa is comparable to that with fresh spermatozoa.
The benefits of spermatozoa cryopreservation are obvious. First, by cryopreserving excess sper- matozoa, the male partners do not have to go through repeated sperm retrieval procedures for another ICSI cycle. Second, synchronization of spermatozoa retrieval and oocyte retrieval is not necessary, thus easing the physician’s and the couple’s tension. Third, collection and cryopre- servation of spermatozoa at the diagnostic procedures or at microsurgery (e.g. vasectomy reversal) allows for future ICSI cycles.
Table I. Comparison of frozen–thawed and fresh PESA sperm for couples undergoing ICSI
Frozen–thawed Fresh p-value
No. of cycles 96 17 Mean no. of oocytes retrieved 14.9 4.5 15.2 4.7 NS Mean no. of injected oocytes 11.0 2.9 11.3 3.4 NS Fertilization rate (%) 71.6 69.2 NS Cleavage rate (%) 89.8 90.2 NS Mean no. of embryos transferred 3.8 1.1 4.0 1.3 NS Implantation rate (%) 14.0 13.2 NS Clinical pregnancy (n, %) 39 (40.6) 7 (41.2) NS No. of deliveries (n, %) 33 (34.4) 6 (35.3) NS
Table II. Clinical pregnancies in each attempt of ICSI
First Second Third Fourth
No. of cycles 51 28 16 1 No. (%) of clinical pregnancies 21 (41.2) 11 (39.2) 6 (37.5) 1 (100)
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Several studies have demonstrated that fresh and frozen–thawed epididymal spermatozoa obtained by MESA produced similar fertilization and pregnancy rates (7–9,12). Similar studies on PESA-spermatozoa are scarce, and there was concern that PESA might not provide enough spermatozoa for cryopreservation (9,10,13). Until now there have been only five studies describing cryopreservation of PESA-derived spermatozoa (Table III). Our study, having the largest cycle number to date, compares favorably with the other studies. This study confirms that fresh and frozen–thawed epididymal spermato- zoa obtained by PESA produce comparable results with ICSI.
The best epididymal sperm retrieval method is controversial. We prefer PESA because it is less invasive, less expensive, easier to learn, and asso- ciated with fewer complications. PESA has been shown to be as effective as MESA with compar- able pregnancy rates (18). Friedler et al. demon- strated that the clinical outcome of ICSI with fresh and frozen–thawed spermatozoa after retrieval by PESA was similar to that by MESA (11). In view of its clinical advantages and pro- viding a similar outcome to MESA, we consider that PESA in combination with ICSI should be the treatment of choice for patients with obstruc- tive azoospermia. This study also demonstrates that PESA is an effective sperm retrieval proce- dure in that PESA could obtain sperm in 91.1% of cases and after freeing–thawing motile sperma- tozoa could be found in 96.1% of cases.
In this study, a mean of 6.5 cryotubes could be stored from each successful PESA procedure to provide more than one ICSI cycle. As shown in Table II, 28 of the cycles were second cycles and 16 were third cycles. One woman even gave birth to four babies after three pregnancies resulting from three ICSI cycles. Only two other studies have mentioned the number of vials of sperm cryopreserved from PESA. Hutchon et al. obtained a mean of 3.77 straws of sperm for cryopreservation by PESA (14). Friedler et al.
found no significant difference in the mean num- ber of vials of spermatozoa cryopreserved follow- ing MESA or PESA (12.8/patient by MESA, 10.6/patient by PESA) (11). These data imply that PESA not only provides sufficient sperma- tozoa for cryopreservation but also allows for several ICSI cycles.
The freezing–thawing process does impair the quality and the motility of spermatozoa. Cayan et al. demonstrated that cryopreservation caused a significant reduction in motility of epididymal spermatozoa, from 20% to 9% (12). Holden et al. found that the motility of epididymal sperm decreased from 13% to 1% after freezing and thawing (7). The detrimental effect of freezing and thawing on sperm motility was also observed in our study. Mean sperm motility decreased from 15.2% to 4.2%, and in two patients (3.9%), cryopreserved samples showed total immotility after thawing. The prefreeze…