IVF Unit, Department of Obstetrics and Gynecology, Hadassah-Hebrew University Medical Center, Ein-Kerem, Jerusalem, Israel
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Abstract |
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Key words: fine needle aspiration/ICSI/non-obstructive azoospermia
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Introduction |
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Materials and methods |
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Oocyte retrieval and insemination
Ovarian stimulation included a long protocol of gonadotrophin-releasing hormone (GnRH) agonist, using nafarelin acetate (Synarel, Teva, Petah-Tikva, Israel) 600 mcg/day by nasal spray, from the 21st day of the cycle, and human urinary follicle stimulating hormone (uFSH, Metrodin, Teva), started 14 days later. Oocyte retrieval was performed 3436 h after human chorionic gonadotrophin (HCG; Chorigon, Teva) was administered i.m., under transvaginal ultrasound guidance and sedation-analgesia. Oocytes were incubated in human tubal fluid (HTF) medium, supplemented with 7.5% synthetic serum supplement (SSS; Irvine Scientific, Santa Ana, CA, USA). In preparation for ICSI, the surrounding cumulus cells were removed 2 h after oocyte retrieval using hyaluronidase (Type VII, Sigma, St. Louis, MO, USA), 60 IU/ml. ICSI was performed on the heated stage of an inverted microscope (Nikon Diaphot; Nikon, Tokyo, Japan), equipped with Leitz mechanical manipulators. Ready-made injection (Swemed, Uppsala, Sweden) and holding pipettes (Cook, Brisbane, QL, Australia) were used. The injection dish included three drops of the final sperm suspension, three drops of 10% polyvinylpyrrolidone (PVP; Sigma) and three drops of the injection medium (HTFHEPES with 6% SSS), all covered with pre-equilibrated paraffin oil (Sigma). To facilitate the extraction of sperm cells, as the sperm pellet drops were engorged with blood cells and cell debris, the injection pipette was filled with PVP prior to sperm aspiration into the pipette. The spermatozoa recovered were rinsed and immobilized in the PVP drop and directly injected into the oocytes. Oocytes were then placed in drops of HTF7.5% SSS for further incubation.
Sperm collection and preparation
TEFNA was performed immediately following oocyte retrieval under light general anaesthesia. The scrotal area was cleaned with 0.5% chlorohexidine solution and sterile draped. Standing to the left of the patient, the testis was grasped with the fingers of the left hand while the butterfly needle was held between the thumb and index fingers of the right hand. A single gentle slow pass in and out puncture and aspiration was performed in the caput of the epididymis on each side followed by immediate microscopic search of the aspirate to rule out the presence of spermatozoa. In the absence of spermatozoa in the epididymal aspirate, multiple punctures (mean of 15, range 1020 per testis) and aspirations were performed systematically throughout the whole testis on both sides while squeezing and holding the testis firmly in position between the operators fingers throughout the entire aspiration, so that the aspirated locations are clearly plotted. Every testicular puncture was composed of multiple in and out movements in all directions until the yellowish fluid so aspirated ceased to flow or if bloody fluid appeared. The tubing was then occluded with an artery forceps and the needle removed from the testis. The operation came to an end once no more testicular fluid was aspirated or when all the aspirates started to be bloody. The set-up for both epididymal and testicular punctures included 23 gauge butterfly needles connected to a 20 ml syringe, installed in an aspiration handle (Cook, USA) for the application of a steady negative pressure. Following each puncture, the needle end of the tube was cut with scissors and the tube's contents rinsed using a 20 ml syringe filled with HTFHEPES medium (Irvine Scientific) supplemented with 0.4% human serum albumin (Sigma). Medium (~0.5 ml) was flushed into one well of a 4-well plate (Nunc, Copenhagen, Denmark). A new butterfly needle was used for each puncture and punctures continued as long as yellowish, non-bloody fluid continued to flow into the tubing. The testicular aspirates were examined after every four punctures filling a 4-well plate under an inverted microscope (Nikon Diaphot) at x200 magnification and whenever sperm cells were noted, further aspirations in the same testicular area were performed. Samples from the wells in which sperm cells were observed were all collected before centrifugation into a common 15 ml conical test tube (Falcon, Becton Dickinson, Lincoln Park, NJ, USA) while all the other wells in which sperm cells were not observed in the initial inspection were collected in another 15 ml conical test tube. Both tubes were then centrifuged at 1800 g for 5 min. The final sperm suspension was achieved by resuspending the pellet in 50 µl of HTF7.5% SSS. Erythrocyte lysing buffer (Nagy et al., 1997) was used in a few cases when the pellet was contaminated with red blood cells to the extent of not being able to search for sperm cells. Finally, microdroplets of 2 µl were placed on a dish under mineral oil for the microscopic search at x200 to x400 magnification.
Embryonic development and transfer
Fertilization was assessed 18 h after ICSI and confirmed by the detection of two clearly distinct pronuclei (2PN). Embryonic development was evaluated 24 h later. The embryos were scored for quality according to the homogeneity of the blastomeres and the degree of anucleated fragments (Staessen et al., 1989). Embryo transfer was performed 4872 h after oocyte retrieval, using a Casmed catheter (Casmed, Banstead Surrey, UK), loaded with 1015 µl culture medium. Supplementary embryos were cryopreserved. Micronized progesterone in the form of vaginal tablets (Dizengoff Pharmaceuticals, Tel Aviv, Israel) 50 mg, twice daily, were used to support the luteal phase, supplemented, when needed according to serum 17ß-oestradiol and progesterone concentrations on days 4, 8 and 12 following embryo transfer, with i.m. injections of HCG (Chorigon; Teva), 2500 IU.
Statistical analysis
Statistical analyses were performed using Fisher's exact test.
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Results |
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Excess testicular spermatozoa were cryopreserved in 27 cycles and later thawed and used for fertilization in 22 cycles so far. In 18 cycles, embryos were available for transfer although in two cycles the embryos failed to progress and were not transferred. Three pregnancies (18.7%) were achieved following 16 transfers, resulting in the births of two healthy girls and one ongoing pregnancy.
Following TEFNA, patients were released after 25 h, resuming their normal activities. No haematoma, infection or other serious side-effects were reported following TEFNA. The only complaints were pain that lasted 3 days in three patients and treated with oral analgesics after physical examination, testicular Doppler ultrasound and blood tests were normal.
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Discussion |
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Our study did not compare the efficacy of testicular sperm retrieval by FNA with that of TESE by open biopsies. Nevertheless, comparisons between open and percutaneous needle biopsies in men with azoospermia (Rosenlund et al., 1998) have shown that needle biopsies using 19 gauge needles were inferior to open biopsy for histopathological evaluation, but when testicular material was obtained, it was as good as open biopsy in terms of determining the presence of spermatozoa. The results with 21 gauge needles were inferior to open biopsy in both parameters. The authors concluded that percutaneous biopsy with a 19 gauge butterfly needle is a quick and reliable method for demonstrating spermatozoa for ICSI. In another study (Friedler et al., 1997
) spermatozoa were recovered in 16 out of 37 patients (43%) by open biopsies, compared to four of 37 patients (11%) by TEFNA. Such poor results may be related to the limited number of punctures, as only six were performed in one testicle. Furthermore, the multiple-step sperm preparation including Percoll gradient separation and two additional washing steps described by this group is unsuitable, in our opinion, for this purpose as it is bound to cause loss of spermatozoa during the various steps and when only few are present this may result in failure of sperm recovery. The preparation protocol we use for testicular spermatozoa includes only one step of high-speed (1800 g for 5 min) centrifugation and the search takes place in 2 µl microdroplets.
Although the ultimate choice of testicular sperm retrieval methods will depend on a number of factors including the clinical diagnosis, patients preference and the availability of the necessary surgical skills, one of the most important parameters will be the physiological consequences of these techniques on testicular function. In a recent study (Schlegel and Su, 1997), the effects of TESE on the testis of 64 patients were evaluated. Of patients, 82% had intratesticular abnormalities present on ultrasound, suggestive of persistent haematoma and/or inflammation as long as 3 months following TESE. The majority of these lesions were transient and appeared to resolve by 6 months after the operation. However, permanent devascularization of the testis was shown to occur following TESE procedures with multiple biopsies. Another study (Harrington et al., 1996
) demonstrated sonographic evidence of intratesticular bleeding in four out of 58 (7%) percutaneous biopsies performed with an 18 gauge biopsy needle, resolving within 6 months post-operatively. On the other hand, in the study by Watkins and co-workers who applied fine needle biopsy, as well as in our study, no major complications were recorded, beside mild pain and discomfort requiring simple analgesics (Watkins et al., 1997
). In the present study, a very low rate of minor complaints was recorded (3/85; 3.5%), all three cases presenting only pain that was treated by oral analgesics after physical examination, testicular Doppler ultrasound and blood count were normal. However, a comparative analysis on the long-term effects of these techniques as revealed by extensive sonography is warranted.
Following TEFNA we were also able to cryopreserve spare testicular spermatozoa in 27 cycles and three pregnancies (18.7%) were achieved so far following 16 transfers, resulting in the births of two healthy girls and one ongoing pregnancy. This is in agreement with a previous observation (Verheyen et al., 1997) which found that, despite the low quality of the fresh testicular spermatozoa, a high percentage of frozenthawed testicular spermatozoa survive and are capable of successfully fertilizing mature oocytes.
Finally, our results support the conclusion of previous studies that testicular sperm search should be offered to all azoospermic patients, irrespective of FSH concentrations, testicular size, medical history and testicular histology (Tournaye et al., 1995, 1996a
, 1997
; Kahraman et al., 1996a
,b
). Our results also demonstrate high rates of sperm recovery in patients with Sertoli cell-only or maturation arrest, where previous diagnostic histology failed to demonstrate the presence of mature spermatozoa. This fact underlines the shortcomings of a single, or even several testicular specimens for making a correct histological diagnosis and emphasizes, in our opinion, the focal nature of spermatogenesis in these patients. This concept of focal distribution was challenged recently by Silber et al., who postulated that a rather homogeneous distribution of spermatogenesis exists in non-obstructive azoospermia (Silber et al., 1997
). Nevertheless, we have documented that in most cases where only a few spermatozoa were recovered, these were observed in only one or two of many wells of the fresh preparation examined on-line for the presence of spermatozoa, while all other wells were devoid of spermatozoa.
In conclusion, TEFNA was shown to be a successful approach for collecting mature spermatozoa in the majority of cases with non-obstructive azoospermia. The technique is easy to learn and allows the operator the possibility of reaching multiple intratesticular sites and increases the chances of retrieving spermatozoa for ICSI, almost without reduction in testicular volume. The procedure was also found to be safe and well tolerated by all patients. In our opinion, until exclusion criteria are defined, no case should be considered hopeless and refused a testicular sperm recovery attempt, and TEFNA should be considered the first choice for sperm recovery attempts.
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Notes |
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References |
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Submitted on August 19, 1998; accepted on March 11, 1999.