In the past decade, the techniques of epididymal sperm retrieval and micromanipulation to assist fertilization have been two of the most exciting developments in the field of male infertility treatment. Men with congenital bilateral absence of the vas defences (CBAVD) or surgically unreconstructable reproductive tract obstruction are now able to achieve pregnancies with use of these advanced techniques.
We have found that microsurgical epididymal sperm aspiration (MESA) using a glass micropipet technique simultaneous to intracytoplasmic sperm injection (ICSI) appears to maximize opportunities of pregnancy for these infertile couples with unreconstructable male reproductive tract obstruction.
MESA from individual epididymal tubules with a micropuncture technique allows retrieval of high numbers of sperm with optimal quality for immediate use during ICSI as well as for cryopreservation. A unique micropuncture pipet holding apparatus, MESA- Holder has been developed and patented at Cornell. Its unique 180° angle adjustable pipet holding system simplifies the procedure of micropuncture epididymal sperm retrieval. The micropuncture technique is an atraumatic technique that limits damage to the epididymal tubules and avoids potential blood cell contamination of the epididymal fluid, while yielding high quantities of motile spermatozoa.
Effects of Chronic Reproductive Tract Obstruction
The effect of chronic reproductive tract obstruction on the location and quality of spermatozoa must be appreciated prior to any attempts at sperm retrieval. The site of sperm retrieval affects sperm quality, and the quality of retrieved sperm in turn may affect the chances for successful fertilization and cryopreservation of retrieved sperm.
It has long been thought that sperm exiting the testis lack maturity, motility, and fertilizing capability and that transit through the epididymis is essential to the acquisition of these features. Several investigators, however, have shown that although in the unobstructed setting sperm quality improves as the spermatozoa travel from caput (head) to cauda (tail)epididymis, this is not true in the obstructed situation. In reproductive tract obstruction, improved motility is seen in sperm retrieved from the caput epididymis compared to the cauda. Sperm aspirated from the efferent ducts and caput epididymis can be morphologically similar to sperm from normospermic men except for the presence of cytoplasmic droplets. In contrast, sperm from the tail of an obstructed epididymis are in advanced stages of degeneration and necrosis. Normal sperm are absent or rare, whereas macrophages filled with phagocytized sperm remnants are seen in abundance. In the presence of reproductive tract obstruction, continual sperm production by the testis occurs and ongoing resorption of dead and degenerated sperm in the distal epididymis is found. This finding, referred to by some as "inverted motility", is illustrated in Figure 1.
Thus, although it has long been thought that sperm exiting the testis are immature and incapable of fertilization, this holds true only in the unobstructed system. Therefore, in obstruction, better quality sperm can be found proximally in the efferent ducts or caput epididymis, and the distal epididymis is the site of sperm degeneration. These factors should be taken into account during any attempt at sperm retrieval.
Candidates for MESA: Obstructive Azoospermia
Candidates for sperm retrieval include men with acquired or congenital reproductive tract obstruction, and men who elect to undergo sperm retrieval as an alternative to surgical reconstruction. Acquired reproductive tract obstruction can occur as a result of infection, trauma, iatrogenic injury or previous failed vasovasostomy or vasoepididymostomy. Iatrogenic injury can occur during bladder neck, pelvic, abdominal or inguino-scrotal surgery.
Congenital anomalies of the male reproductive tract are relatively uncommon in the general population, but can occur in up to 2 percent of infertile men. Perhaps the best known congenital anomaly of the male reproductive tract is congenital bilateral absence of the vas deferens (CBAVD) which occurs in almost all men with cystic fibrosis. CBAVD is now regarded as most commonly reflecting a mild form of cystic fibrosis in which only the reproductive tract anomalies are manifest without pulmonary or digestive symptoms. CBAVD can also probably occur as a result of Wolffian duct anomalies without evidence of cystic fibrosis gene mutations. Unilateral vasal aplasia occurs in less than one percent of the male population and can also be associated with cystic fibrosis gene mutations.
In all cases of reproductive tract obstruction, the optimal treatment is one that will allow the couple to conceive with minimal intervention . When this is impossible (as in CBAVD) or has already failed (as in previous attempts at vasovasostomy or vasoepididymostomy) consideration must be given to the use of sperm retrieval directly from the epididymis or testis with assisted reproduction. Preferably, sperm should be retrieved and cryopreserved during attempts at reconstruction. Because of the association of CBAVD with cystic fibrosis mutations and Wolffian duct anomalies, all men with CBAVD should have renal ultrasounds and their wives should have cystic fibrosis gene mutation analysis prior to attempts at fertility.
Although reports on microsurgical epididymal sperm aspiration with IVF appeared in the literature as early as 1984, the first live birth was not reported until 1990. As it was first described, the procedure of microsurgical epididymal sperm aspiration involved careful dissection of the epididymis under the operating microscope and incision of a single tubule.
Once incised, fluid spills from the epididymal tubule and pools in the epididymal bed. This pooled fluid is then aspirated. Because the epididymis is richly vascularized, this technique invariably leads to contamination by blood cells that may affect sperm fertilizing capacity in vitro.
Our initial experience with this "pool and aspirate" technique was unsuccessful due to contamination of the aspirated sperm with blood products. This appeared to result in impaired sperm function and the inability to fertilize oocytes in vitro. For this reason we developed a technique of micropuncture of the epididymal tubule to avoid blood product contamination. This technique, combined with improved ovarian stimulation techniques and micromanipulation of retrieved sperm, has resulted in markedly improved fertilization and pregnancy rates in our patients with unreconstructable reproductive tract obstruction. The technique of microsurgical epididymal sperm retrieval offers the advantages of minimizing contamination of epididymal fluid with bloodcells, repeated aspirations can be performed, and aspiration of sufficient quantities of fluid for immediate use as well as for cryopreservation are possible.
(1) Aspiration Device
In order to aspirate fluid from within the epididymal tubules a device is needed that is sharp and fine enough to be able to pierce the tubule successfully, and avoid the extensive, delicate network of Vessels that cover the epididymis. To achieve these goals micropipettes with tip widths of 250 to 350 um were hand drawn from glass tubing with an outer diameter of 0.9 mm and inner diameter of 0.6 mm, and then hand sharpened on a grinding wheel. The micropipette is then attached to silicone tubing and a three-way stopcock. Two syringes are attached to the stopcock, a 1 cc tuberculin syringe and a 10 cc glass syringe. The tuberculin syringe collects the epididymal fluid when sufficient fluid is obtained, while the glass syringe provides fine control of the aspiration as well as rapid equilibration of pressure to avoid aspiration of blood outside of the epididymal tubules (Figure 2).
A unique micropuncture pipet holding apparatus, MESA- Holder has been developed and patented (Schlegel, Li and & Goldstein) at Cornell (Figure 3). Its unique 180° angle adjustable pipet holding system simplifies the procedure of micropuncture epididymal sperm retrieval. The micropuncture technique is an atraumatic technique that limits damage to the epididymal tubules and avoids potential blood cell contamination of the epididymal fluid, while yielding high quantities of motile spermatozoa. In this system it is imperative that the epididymal fluid never contact the glass syringe as the sperm may adhere to the glass surface.
(2) Operative Procedure
The patient is explored through a midline scrotal incision. The testis is delivered and tunica vaginalis is opened to expose the epididymis (Figure 4).
The operating microscope is brought into the sterile field and the epididymis is examined under 8 to 15X magnification. The obstructed epididymis has a characteristic appearance. The tail of the epididymis has dilated yellow tubules due to the predominance of macrophages and degenerating sperm. The first puncture for aspiration is made proximal to these yellow tubules. If the tubules can be clearly visualized with the epididymal tunic intact, the puncture can be made through the tunic (Figure 5).
If however the tubules are obscured by the tunic, a linear opening in the tunic is made with a 15 degree microknife after coagulating the surface with the bipolar electrocautery. An alternative approach is to incise tubules and gather fluid after it flows out of the tubules.
With the assistant stabilizing the testicle, a suitable tubule is punctured by the operating surgeon and fluid is gently aspirated (Figure 6). The assistant can facilitate retrieval by gently compressing the testis and epididymis. When there is no longer flow, the retrieved fluid is back flushed through the system using 0.5 cc of human tubal fluid and handed to the in vitro fertilization team who are standing by in the operating room.
They examine the fluid immediately under the microscope to assess sperm count and motility. Sequential micropunctures can be performed until optimal sperm quality has been obtained. Typically approximately 100 x 106 sperm with good motility are retrieved using this MESA approach. Because sperm in the epididymal fluid are highly concentrated (roughly 1 x 106 /ul), only microliters quantities of epididymal fluid are needed to be retrieved. In this way, MESA provides for more than adequate numbers of sperm for immediate use with ICSI, as well as for sperm cryopreservation. (Figure 7)
If optimal sperm quality has not been found in the caput or body of the epididymis, fluid can be retrieved from the efferent ductules with the micropuncture technique. The efferent ductules arise from the superior pole of the testis, just superior to the testicular vessels. Therefore, with the assistant orienting the testicle properly, the operating surgeon can expose the efferent ductules by incising the tunic at the junction of the epididymis and testis. In this way the caput epididymis is dissected bluntly off of the testis. After adequate fluid has been retrieved the puncture sites are closed with 10-0 monofilament nylon sutures. The tunic incisions are closed with 6-0 polypropylene or larger. The tunica vaginalis is closed in a water tight fashion to avoid inflammation and adhesions that could complicate future explorations. Epididymal fluid samples are taken from the operating room to the in vitro fertilization laboratory where they are subjected to mini-Percoll discontinuous gradient centrifugation, swim-up, and/or sedimentation to remove debris, macrophages and blood products.
(1) Percutaneous epididymal sperm aspiration
In addition to retrieving sperm from the epididymis through open microsurgical techniques, percutaneous epididymal sperm aspiration (PESA) is also possible. The advantages to this technique are that it can be performed without surgical scrotal exploration, it can be repeated easily and at low cost, and it does not require an operating microscope or expertise in microsurgery. The procedure as described by Craft et al. has been performed under local or general anesthesia. After induction of anesthesia, the testis is stabilized and the epididymis is held between the surgeon's thumb and forefinger.
A 21-gauge butterfly needle attached to a 20 ml syringe is inserted into the caput epididymis and withdrawn gently until fluid can be seen entering the tubing of the aspiration set (Figure 8).
The tubing is clamped, the 20 ml syringe removed, and the tubing is back flushed with medium. The procedure is repeated until adequate amounts of epididymal fluid with motile sperm are retrieved1. If no sperm are retrieved, as occurs in at least 20% of sperm retrieval attempts, then it is necessary to proceed with MESA, testis biopsy or testicular aspiration.
(2) Percutaneous testicular sperm aspiration
The procedure for percutaneous testicular sperm aspiration is similar to PESA. The procedure has been performed under general or local anesthesia. After induction of anesthesia, the testis is stabilized between the surgeon's thumb and forefinger with the epididymis oriented posteriorly. A 22- or 23-gauge one and one half inch needle attached to a 20 ml syringe is inserted along the long axis of the testis from the inferior pole directed toward the superior pole. The needle is withdrawn slightly and redirected several times in order to disrupt the testicular architecture so that seminiferous tubules can be aspirated. The procedure is repeated until adequate amounts of testicular material are retrieved. The procedure is depicted in Figure 6. For optimal retrieval, a syringe holder is used to provide significant negative pressure for the aspiration. The Franzen hand-grip syringe holder accommodates one disposable syringe and allows aspiration to be performed with one hand while the other hand stabilizes the testis. The Franzen syringe holder is depicted in Figure 9.
Although only sporadic reports of fertilization and pregnancy rates achieved with percutaneously aspirated testicular sperm exist, there are reports on success rates with sperm retrieved from open testicular biopsy and manipulated with IVF/ICSI. One such study reported fertilization and clinical pregnancy rates of 45 and 62 percent for epididymal sperm compared to 46 and 42 percent for testicular sperm, respectively. The ongoing pregnancy rates were 50 and 43 percent for sperm retrieved from the epididymis and testis, respectively. Testicular sperm can also be recovered using fine needle aspiration ( FNA), percutaneous biopsy, or an open technique of testicular sperm extraction (TESE).
The superior success rates with MESA and IVF/ICSI can be ascribed to several factors. First, the experience of the centers performing IVF/ICSI plays a large role. The centers that have had the greatest experience with MESA and IVF/ICSI also have demonstrated consistently outstanding results for IVF/ICSI using ejaculated sperm. By direct injection of sperm into the oocyte cytoplasm, ICSI has allowed for even severely abnormal ejaculated sperm to fertilize and achieve pregnancy. Secondly, the microsurgical technique is very important to the success of MESA. Microsurgical epididymal sperm aspiration allows for meticulous hemostasis during retrieval and therefore minimal contamination of epididymal fluid with blood cells. In addition, microsurgical aspiration allows for more than adequate amounts of sperm to be retrieved per aspiration procedure so that in addition to processing a portion immediately, sperm may be preserved for later cycles limiting the patient to one procedure for sperm retrieval.
Experience with the last 81 couples with obstructive azoospermia who selected to undergo MESA-ICSI at our institution from March, 1995 to April, 1998 at Cornell (Schlegel at el.) is presented. Sperm was retrieved from the epididymis in 81/81 (100%) attempts, despite multiple aliquots of previous unsuccessful sperm retrieval attempts at other institutions. In all 81 cases motile sperm were cryopreserved as well. Clinical pregnancies were achieved in 76%(62/81) cycles for these couples. Ongoing pregnancies or deliveries have occurred for in 65% (53/81) cycles of simultaneous MESA-ICSI.
Simultaneous MESA-ICSI appears to provide optimal pregnancy and delivery rates for couples where the man has unreconstructable reproductive tract obstruction
Table 1. MESA-ICSI: Obstructive Azoospermia
|(Schlegel et al. NYH-Cornell)||Date of MESA Procedure|
|8/93 - 2/95||3/95 - 4/98|
|Fertilization Rate||172/336 (51%)||744/918 (81%)|
|Clinic Pregnancy Rate||17/37 (46%)||62/81 (76%)|
|Ongoing/Delivered||15/37 (41%)||53/81 (65%)|
1. The location of optimal sperm quality in obstructed men differs from unobstructed men in that motile sperm capable of fertilization can be retrieved from the more proximal portions of the epididymis, and efferent ducts, whereas the distal obstructed reproductive tract is the site of sperm degeneration.
2. The micropuncture technique of epididymal sperm aspiration has the advantages of minimal contamination by blood cells, meticulous hemostasis, and the ability to retrieve ample amounts of fluid for immediate use and for cryopreservation.
3. Of the sperm retrieval and assisted reproductive techniques, the most successful combination reported to date is microsurgical epididymal aspiration with intracytoplasmic sperm injection. Ongoing pregnancy and delivery rates of 48 percent or more per attempt at sperm and egg retrieval are currently achieved at experienced centers.
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