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From the * Division of Urological Surgery,
Washington University School of Medicine, St. Louis, Missouri; and the
Department of Anatomic Pathology and
Glickman Urological Institute, The Cleveland
Clinic Foundation, Cleveland, Ohio.
| Correspondence to: Cathy K. Naughton, Assistant Professor, Division of Urologic Surgery, Director of Male Infertility and Microsurgery, Washington University School of Medicine, Infertility and Reproductive Medicine Center, 4444 Forest Park Avenue, Suite 3100, St. Louis, MO 63108 (e-mail: naughtonc{at}msnotes.wustl.edu). |
| Received for publication December 2, 2003; accepted for publication March 2, 2004. |
| Abstract |
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Key words: DMSO, vas deferens, testicle, epididymis
The concept of an intravasal method for occlusion with the option of subsequent reversal is not new. Earlier attempts to develop reversible occlusive technology focused on rigid and flexible prosthetic devices (Brueschke et al, 1974a,b,c, 1975, 1979, 1980). These devices require surgical implantation, cause vasal wall erosion, and are unable to reliably occlude the vas deferens. More recent technologies have focused on the less invasive percutaneous intravasal delivery of materials, including silicone rubber, an elastomeric-hydrogel matrix plug, and medical polyurethane (Carroll et al, 1993; Soebadi et al, 1995). Although these techniques provide relatively good occlusion, the materials demonstrate significant histological effects on tissues (Chen et al, 1996). Furthermore, the reversibility of these agents is unknown. A recent report on styrene maleic anhydride used as an occlusive agent and dimethyl sulfoxide (DMSO) as a reversal agent was studied in langar monkeys (Lohiya et al, 2000). Vasal occlusion causing uniform azoospermia required 2 injections, and reversal required a relatively labor-intensive technique (palpation, percutaneous electrical stimulation, forced vibratory movement, suprapubic percussion, and per-rectal digital massage of the vas segments).
Efforts to develop less invasive techniques for "permanent" vasectomy with the option of easy "reversibility" has led to the possible application of a chemical occlusive agent, URYX (Genyx Medical, Inc, Aliso Viejo, Calif). URYX is an expansion of the product EMBOLYX (Micro Therapeutics, Inc, San Clemente, Calif), a biocompatible polymer of ethylene vinyl alcohol. This polymer is dissolved in a DMSO carrier to allow the injection of a very low viscosity fluid into tissues or a vessel. Once the dissolved material comes into contact with body tissue or fluids, the DMSO rapidly dissipates from the polymer, which results in a precipitate of a coherent solid mass (Figure 1). Success has been achieved with EMBOLYX (Micro Therapeutics) for the interventional embolization of vascular malformations in swine rete mirabile, for tumor control by selective arterial occlusion in a rabbit model, and in human clinical cases of brain arteriovenous malformations (Murayama et al, 1998; Chaloupka et al, 1999; Nahser et al, 1999; Wright et al, 1999). URYX has been successfully used for selective fallopian tube occlusion in a rabbit model. Pregnancy was subsequently confirmed in the nonembolized fallopian tube in 9 of 10 rabbits receptive to breeding. No pregnancies were observed on the embolized tubes. There was no gross evidence of inflammation, adhesions, or URYX in the peritoneum in any animal (Abdala et al, 2001).
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The presence of URYX grossly causes pigmentation within the fallopian tubes but, histologically, there is only mild to moderate inflammation and fibrosis (Abdala et al, 2001). DMSO alone causes a transient reduction in renal artery blood flow when it is injected intra-arterially, but it does not cause obstruction of flow (Murayama et al, 1998). Histologically, kidneys exposed to DMSO alone demonstrate moderate to marked multifocal acute mural necrosis in larger blood vessels (Wright et al, 1999). When DMSO is rapidly injected (5 seconds) into the swine rete mirabile, macroscopic examination of the brain shows acute subarachnoid hemorrhage around the brain stem. Microscopically, there is extensive transmural fi-brinoid necrosis of the vascular walls and rete vessel occlusion by faintly eosinophilic material containing nuclear debris. In contrast, in a 15-second slower injection of DMSO, only focal mild inflammation is observed (Murayama et al, 1998). The safety and histological effects of URYX and DMSO to the vas deferens, epididymis, and testis are unknown. The purpose of the present study was to determine, in a rabbit model, whether URYX is safe and effective in causing occlusion of the vas deferens and whether subsequent patency can be achieved by dissolving URYX using its organic solvent, DMSO.
| Materials and Methods |
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Vasal Surgery![]()
The bilateral vasa were exposed in a similar fashion in vasectomy, URYX
injection, and DMSO reversal. All procedures were performed by a single
surgeon (C.K.N.) who used 2.5x magnification loops. Animals were
anesthetized with an intramuscular injection of xylazine (4 mg/kg) and
ketamine (50 mg/kg) and a subcutaneous injection of acepromazine (1.0 mg/kg).
A 1-cm suprapubic transverse incision was made in the midline one
fingerbreadth cephalad to the pubic symphysis. The spermatic cords were
brought up through the incision and isolated. The cremasteric fascia was
incised in a longitudinal fashion, and the vas deferens was isolated with its
blood supply, using a towel clip.
For those animals undergoing vasectomy, a 1-cm segment of the vas deferens was excised, the lumen was cauterized, and the distal and proximal ends were ligated with 6-0 Prolene sutures. The excised segment of vas deferens was placed in formalin and processed for histological confirmation.
For those animals undergoing URYX injection, the vas deferens was cannulated with a 30-gauge lymphangiographic needle (003401; Cook Urological Inc, Spencer, Ind), which had been preflushed with DMSO, just distal to the convoluted vas deferens. Vortexed URYX, which contained micronized tantalum powder added to the polymer/solvent mixture to obtain radiopacity, was slowly injected (0.05 mL/30 seconds) intravasally. The URYX was allowed to solidify for 1 minute (Figure 2). The needle was removed, and the vasal muscularis at the site of injection was tagged with a 6-0 Prolene suture. Intraoperative fluoroscopy was used to document successful URYX injections (Figure 3a).
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For animals undergoing DMSO reversal, intraoperative fluoroscopy was used to document the status of the URYX plug after 1 month of biweekly ejaculations (Figure 3b). The vas deferens was exposed, and the site of the previously placed Prolene suture was isolated. The DMSO preflushed 30-gauge lymphangiographic needle was used to cannulate the lumen of the vas deferens and to inject 1.5 mL of DMSO intravasally proximal to the Prolene suture. After all procedures, the skin incision was closed using interrupted 4-0 nylon sutures. The treatment of vasal units in E1 and E2 is shown in Table 1.
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Semen Analyses![]()
Pretreatment and postprocedure semen was collected biweekly using a
"homemade" artificial vagina described elsewhere
(Naughton et al, 2003). Collected ejaculates were analyzed for volume, total sperm count, sperm
density, and motility. Azoospermia was confirmed by examining 20 fields of a
stained smear pellet at 200x magnification after centrifugation at 800
x g for 7 minutes. All semen analyses parameters were
determined manually by one investigator (C.K.N.).
Histological Analyses![]()
Animals were sacrificed 1 month after the vasectomy and URYX-only injection
procedures (E1). Those animals that underwent subsequent DMSO reversal
attempts were sacrificed 1 month after the DMSO injection (E2). All animals
were sacrificed with an intravenous overdose of Beuthanasia-D (Schering-Plough
Animal Health, Kenilworth, NJ). Testicles, epididymides, vasa, and vasal
ampulla were removed en bloc and placed in formalin for histological
examination. The distal vas, injection site, proximal vas, cauda epididymis,
caput epididymis, and testis were each examined separately in paraffin blocks.
Blocks were sectioned at 6 microns and stained with hematoxylin and eosin.
Tissues were examined for the presence of URYX and assessed for signs of
inflammation. The inflammatory response of the wall and adventitia of the vas
deferens was given a score (015) that was based on the sum of
semiquantitative grades (0 = none, 1 = mild, 2 = moderate, and 3 = severe) for
the following categories: foreign body giant cell reaction, granulation
tissue, lymphocytes, eosinophils, and scarring. Inflammatory response scores
of 15, 610, and 1115 corresponded to mild, moderate, and
severe changes, respectively. The histological evaluation of tissues was
performed blindly by a single pathologist (J.M.).
| Results |
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Pathology End Points![]()
Before animals were sacrificed and their tissues subsequently harvested, 3
vasal units were subjected to an injection of URYX only, 7 vasal units were
subjected to vasectomy, 3 vasal units were subjected to injections of URYX
then NS, and 3 vasal units were subjected to injections of URYX then DMSO
(Table 3). There was no
significant difference in vasal inflammatory response scores between vasal
units treated with URYX only, compared with vasal units that underwent
vasectomy. A mild inflammatory response was observed in the vas deferens of
vasal units treated with URYX alone or with vasectomy. The inflammatory
changes in the vasectomy units were found more in the distal and proximal vas
adventitia, when they were present. Vasitis nodosum was observed in 4 of 7
vasal units after vasectomy. This was characterized by an obliterated vasal
lumen and moderate vasal adventitia inflammation
(Figure 4c). The other 3 vasal
units that had undergone vasectomy did not contain the area of vasal excision.
In contrast, the vasal units subjected to injection with URYX only did not
show proximal vasal inflammatory changes, but these changes were observed at
the injection site and distal vas. Vasal units subjected to injection with
URYX followed by DMSO demonstrated the greatest inflammatory response scores,
compared with vasal units treated with URYX followed by NS, URYX alone, or
vasectomy alone. A moderate inflammatory response was particularly evident in
the vasal lumen at the injection site
(Figure 4aj).
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Epididymal and testicular histology remained unaffected in all vasal units from the E1 group. Those vasal units subjected to injection with URYX followed by DMSO in the E2 group, however, showed evidence of adhesions, necrosis, and degenerating cells in the epididymis and foreign body giant cell reaction in the testis (Figure 4m, n). In contrast, the vasal units in the E2 group subjected to injection with URYX followed by NS showed no histological abnormalities of the epididymis and testis (Figure 4k, l), comparable to the epididymides and testes of control animals that underwent bilateral vasectomy only.
| Discussion |
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Our initial experiments were aimed at determining how much URYX is required for complete vasal occlusion. We determined that the unilateral injection of 0.1 mL of URYX and contralateral vasectomy is sufficient to cause azoospermia after 2 to 3 ejaculations, and this persisted up to 1 month. We then injected 0.1 mL of URYX bilaterally and determined that azoospermia was achieved within 1 to 3 ejaculations, similar to control animals that underwent bilateral vasectomy (Table 2). After 1 month of biweekly ejaculations, however, the URYX plug was found to have migrated distally towards the ampulla. This was clearly demonstrated by fluoroscopic images obtained before the animals were sacrificed and histologically by the presence of URYX material at the ampulla instead of the straight portion of the vas deferens, where it was initially injected (Figure 4p). Because the longest interval between URYX injection and any animal's death was only 2 months, the long-term effects of the injected material are not known. Furthermore, the ability to reliably reverse vasal occlusion using DMSO when the location of the plug varies is suspect. Histologically, URYX alone did not appear to cause a significant tissue reaction. However, the histological consequences of the technical misplacement of URYX into the vasal muscularis instead of the vasal lumen demonstrated scarring (Figure 4d).
We were not able to demonstrate any reversible effect on URYX by DMSO in
our study. We believe that the most likely reason is the low contact time of
DMSO with the URYX plug. Completely submerging a URYX plug into a solution of
DMSO will result in the liquefaction of the plug
(Figure 1d); however, the URYX
plug in a lumen may not have been exposed to enough DMSO or have had enough
contact time with the solvent to cause the URYX plug to dissolve.
Additionally, the vasal administration of DMSO results in significant vasal,
epididymal, and testicular inflammatory changes
(Figure 4). We believe that
these effects were caused mainly by DMSO, given that no epididymal or
testicular histological abnormalities were found in animals injected with URYX
alone. Our data suggest that the repeated vasal injections may have
contributed to the increased inflammation seen in injected vasal units URYX
and NS, compared with URYX alone. A strong conclusion regarding the
histological end points is difficult to determine because of the small number
of animals used and the variability of inflammatory response among the animals
(Table 3). One animal (36) died
7 days after the injections with DMSO and NS. This animal demonstrated
decreased feeding
23 days after anesthesia for the reversal (DMSO)
procedure. We postulate that the death of this animal was related to
anesthetic/sedative effects rather than to the administration of DMSO. Because
the exact day of death was not known, we elected not to harvest tissue from
this animal, because the interpretation of any tissue histology would have
been affected by the unknown interval of ischemia.
The clinical utility of studying suitable materials for vasal injection is to find a truly reversible vasectomy. The ideal reversible vasectomy would be able to reliably and reproducibly cause vasal obstruction and azoospermia but then to, reliably and reproducibly, easily enable vasal patency to be restored, if desired. At present, vasectomy is a common and reliable method of contraception. A certain percentage of men who undergo a vasectomy will request a reversal. A couple's options for conceiving their own biological child after vasectomy are vasectomy reversal or assisted reproductive techniques, both of which are costly and not always successful.
We have demonstrated, in a rabbit model, that injection with URYX may have the potential for permanent vasal occlusion without significant histological effects in the vas deferens. This material will require more long-term studies in vitro and in vivo before its application to humans. A hurdle in using this material for reversible vasectomy is that the DMSO injected into the vasal lumen will only make contact with a small proximal end of the URYX plug. In vitro testing to explore methods that would increase the exposed surface area and/or contact time of the URYX plug to DMSO is important. Preliminary experiments may be performed using a silicone tube as an ex vivo vas deferens model to determine the time interval and amount of DMSO required to completely dissolve a defined length of URYX plug. This system may be used to determine how the required amount of DMSO will vary as a function of URYX plug length. Future animal studies may require subsequent testing on another animal model, such as the dog, which have vasa more similar to humans than those of the rabbit. Studies in animal models should focus on the significance of URYX plug migration and its consequences on long-term ejaculated semen parameters. The application in humans will, in part, depend on the technical ability of successful intravasal cannulation. On the basis of our data, DMSO is not effective in reversing occlusion created by a URYX plug in the vasal lumen, and it does not appear to be safe for vasal injection in the rabbit model.
| Footnotes |
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