Infertility Clinic, The Family Federation of Finland, Kalevankatu 16, FIN-00100 Helsinki, Finland
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Abstract |
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Key words: intracytoplasmic sperm injection/needle biopsy/pregnancy/testicular biopsy/testicular spermatozoa
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Introduction |
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A simpler fine needle aspiration method was then adopted (Craft and Tsirigotis, 1995), and it has also been used successfully over the last few years (Bourne et al., 1995
; Lewin et al., 1996
; Friedler et al., 1997a
). Testicular needle biopsy, which has been used to obtain biopsy samples for testicular histology (Rajfer and Binder, 1989
; Morey et al., 1994
), is yet another option that can be used to obtain testicular spermatozoa for ICSI (Hovatta et al., 1995
). It has been shown to result in acceptable pregnancy rates in our first small group of patients treated this way.
Cryopreservation of testicular spermatozoa and testicular biopsy specimens is also feasible, resulting in pregnancies after ICSI (Gil-Salom et al., 1996; Hovatta et al., 1996
; Podsiadly et al., 1996
; Friedler et al., 1997b
). Using cryopreservation, repeated invasive procedures can be avoided, but results connected with cryopreservation after needle aspiration or needle biopsy have not yet been reported.
In order to discover the optimal method to carry out testicular sperm retrieval, we analysed the results of our testicular sperm ICSI programme from January, 1994 to September, 1997. We analysed the clinical ICSI parameters and opportunities for cryopreservation, histological diagnosis, and practicality in an in-vitro fertilization (IVF) unit after both needle and surgical biopsies.
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Materials and methods |
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In the beginning of the programme (1994), the biopsies were taken in connection with scrotal exploration, if there was no earlier diagnostic biopsy. After 1995, if diagnosis had not been confirmed earlier by testicular biopsy, needle biopsy was carried out in the clinic prior to the ICSI cycle (nine men). Clinical andrological examinations were carried out in all cases, and the serum concentrations of gonadotrophins and testosterone were measured. A scrotal ultrasound scan was carried out if the testes did not appear normal on physical examination. From 1997, Y chromosome deletions and karyotypes were screened and genetic counselling was given when needed. Prenatal diagnosis was not routinely carried out, but it was available if the couples asked for it.
Four female partners had endometriosis, and ovulatory dysfunction was observed in four others.
Ovarian stimulation and oocyte retrieval
Ovarian stimulation was started by pituitary desensitization with intranasal buserelin (Suprecur; Hoechst, Frankfurt am Main, Germany), 1200 µg daily, from day 23 of the cycle. Stimulation with human menopausal gonadotrophin or follicle stimulating hormone (FSH, Humegon; Oss, The Netherlands; Pergonal or Fertinorm HP; Serono, Geneva, Switzerland) was started, after verifying suppression by an ultrasound scan and serum oestradiol assay. A daily dose of 150300 IU was given until the largest follicles were 18 mm in diameter, at which time an injection of 5000 IU human chorionic gonadotrophin (Profasi, Serono or Pregnyl; Organon) was given. Oocytes were collected by transvaginal ultrasound-guided puncture.
Biopsies
Testicular needle biopsies were performed as described previously by Hovatta et al. (1995). Local anaesthesia with lignocaine around the funicle and to the scrotal skin was used without problems. A biopsy needle (Monopty no.14; Bard Inc, Covington, USA) was inserted under the scrotal skin covering the testis, which was held between the thumb and index finger on one side and the middle finger on the other side, avoiding too much pressure. The needle was then inserted through the tunica, and one to three biopsy cylinders were taken to HEPES-buffered culture medium (Medi-Cult, Copenhagen, Denmark). Histological biopsies were taken to Bouin's fixative. No scrotal haematomas were seen after the needle biopsies and they were not encountered after open biopsies either. Open biopsies were carried out in the operation theatre, either during scrotal explorations under general or spinal anaesthesia, or through a small scrotal incision under local anaesthesia.
Sperm preparation and ICSI procedure
The biopsy samples were taken to culture medium (Medi-Cult or Ciconia IVF Medium; Ciconia Products, Copenhagen, Denmark) and cut into small pieces under a microscope using 25 gauge needles. The sample was then mixed vigorously using a pipette, and the presence of spermatozoa assessed in the cell suspension. The biopsy samples were prepared in a discontinuous two- (40/90%) or three- (40/65/90%) layer Percoll (Pharmacia, Uppsala, Sweden) gradient and resuspended in culture medium if the sperm concentration was regarded to be high enough, or simply pelleted and resuspended in medium without additional washing procedures. Occasionally, the biopsies were carried out one day before oocyte retrieval, as has also been described (Craft et al., 1995), and the samples processed on the day of oocyte retrieval.
The ICSI procedure was performed as previously described (Hovatta et al., 1995). The oocytecumulus complexes were exposed to 80 IU embryo-tested hyaluronidase/ml (Type IV-S; Sigma Chemical Co, St Louis, MO, USA) for <1 min, and then denuded using hand-drawn glass pipettes. The maturation stages of the oocytes were then determined, and metaphase II oocytes were used in ICSI. Before injection, the denuded oocytes were cultured for at least 1 h.
Droplets of testicular cell suspension were placed onto the injection plate, together with two droplets of 5% polyvinylpyrrolidone (PVP; Sigma). A separate 10 µl droplet of HEPES-buffered culture medium for the injections was added to the plate. The plate was covered with embryo-tested filtered mineral oil (Sigma). The injections were performed using Narishige IM-188 and IM-6 micromanipulators (Narishige Co, Tokyo, Japan) on a Nikon Diaphot TMD inverted microscope with Nomarski or Hoffman modulators. Motile spermatozoa were preferentially selected for the injections. The spermatozoa were immobilized, either by aspirating them in and out of the injection pipette or by nicking the tail with a gentle tap of the injection pipette. The oocytes were held by the holding pipette with the polar body at the 6 or 12 o'clock positions, the injection site being at 3 o'clock. After injections the oocytes were washed twice in culture medium and cultured further in Falcon (Becton Dickinson, San Jose, CA, USA) single well dishes, up to six oocytes per dish.
The presence of pronuclei was assessed 1618 h after the injections. Embryos showing two pronuclei and two polar bodies were considered normally fertilized. Their quality was evaluated, and two-embryo transfers were carried out 24 h later. The remaining embryos of good quality were frozen, using propanediol as a cryoprotectant.
Cryopreservation of testicular tissue
For cryopreservation the testicular tissue was cut to small pieces in culture medium. Glycerol (Telko, Espoo, Finland) (7.4%) was used as a cryoprotectant. Glycerol was added slowly drop by drop to the medium containing the biopsy specimens. The biopsy pieces were frozen in 0.5 ml straws which were held for 30 min at 20°C and for 60 min in liquid nitrogen vapour before being placed into the liquid nitrogen.
Statistical analyses
Confidence intervals (95%) of means, the ShapiroWilks test, the two-sample t-test, the MannWhitney U-test and the 2 test were used as appropriate, using SPSS software (SPSS Inc, Chicago, IL, USA).
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Results |
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The mean serum FSH concentration of these six men was 13.6 (10.718.0) IU/l. Four had a histological diagnosis of spermatogenic arrest and two fibrosis.
The mean concentration of FSH among the 14 men with non-obstructive azoospermia was 12.4 (3.544.3) IU/l. It was 14.5 (3.544.3) in the needle biopsy group and 9.9 (4.018) in the open biopsy group. The histological diagnosis among these men were hypospermatogenesis in seven, partial spermatogenic arrest in five and fibrosis of testes in two cases. The man with the highest FSH, 44.3 IU/l, had bilateral cryptorchidism. Spermatozoa were obtained by needle biopsy for ICSI which resulted in a normal pregnancy.
ICSI was carried out in connection with 75 cycles of 51 couples. Spermatozoa obtained using needle biopsy were used in 49 cycles, and those from open biopsy in 29 cycles. In three cycles, spermatozoa from both needle and open biopsies were used for ICSI. The motility of the spermatozoa obtained from the biopsies was evaluated before ICSI, according to World Health Organization guidelines (WHO, 1992). The results are shown in Table I. Grade A motility was seen in only three open biopsy samples and in one needle biopsy sample.
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Two more pregnancies have been achieved after transfer of frozenthawed embryos to women who did not become pregnant after the first embryo transfer.
Histological diagnosis was possible from all 62 diagnostic biopsy samples taken over the same period of time. Nine of these were in connection with patients whose treatment results are included in the present study. In all, 1520 cross-sections of seminiferous tubules per biopsied cylinder of tissue were observed (Figures 1 and 2). The costs of the total treatment procedures, which were calculated during the programme, were 32% lower in the needle biopsy group. The main additional cost of surgical biopsy arose from use of the operation theatre.
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Discussion |
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37 patients with non-obstructive azoospermia were investigated by using both fine needle (21 gauge) aspiration and open biopsy (Friedler et al.,1997a). They obtained spermatozoa by fine needle aspiration in four cases, and by open biopsy in 16 cases. Using needle biopsy, we obtained spermatozoa from all the men with obstructive azoospermia and from six of ten patients with non-obstructive azoospermia. Hence, needle biopsy appears to be at least as good as open biopsy in patients with non-obstructive azoospermia. This is supported by our histological findings, which showed 1520 cross-sections of seminiferous tubules per biopsy specimen. The amount of tissue is sufficient to allow reliable diagnosis and to provide enough spermatozoa for ICSI even in cases of non-obstructive azoospermia. If no spermatozoa are found on immediate examination, two or three biopsy samples can be taken from different directions through the same hole in the tunica albuginea to minimize damage to the testis. This can increase the yield of spermatozoa and minimize the risk of misdiagnosis.
Fine needle aspiration has also been used in the diagnosis of azoospermia (Foresta et al., 1992; Mallidis and Baker, 1994
). It gives either a cytological cell sample (Foresta et al., 1992
), or small fragments of tissue, with full agreement with results obtained using tissue from open biopsy in 56% and slight differences in 36% of the cases (Mallidis and Baker, 1994
). Craft et al. (1997) used 19 and 21 gauge butterfly needles to obtain tissue for histological analysis. They moved the needle in a vertical direction with suction of the tissue, which they cut when removing the needle from the testis. They obtained histological samples from 2025 tubules from 17 men. Using a biopty gun biopsy needle, as in the present study, it is always possible to see the stucture of the testicular tissue in histological examination.
It was also possible to cryopreserve spermatozoa from a needle biopsy sample, as proven by a pregnancy achieved by using spermatozoa from a frozenthawed biopsy sample. Pregnancies have not been reported after using cryopreserved spermatozoa obtained by fine needle aspiration. Although needle biopsy is easy to repeat, it is an invasive procedure which should be avoided whenever possible. The possibility of cryopreservation is one of the advantages of needle biopsy.
Testicular damage after biopsy has been described (Schlegel and Su, 1997). There were avascular areas in the testes after biopsy, probably resulting from rupture of the arteries during the operation. They suggest that open biopsy is safer than needle aspiration because the vessels can be seen during open biopsy. However, in our experience, smaller arteries are not clearly visible and they often lie so close to the tunica that they are cut when the tunica is opened; only the largest arteries can be seen through the tunica. Knowing the anatomy of the testicular artery, it is easy to avoid the main artery under the tunica by taking a needle biopsy sample from either side adjacent to the epididymis. Because the area penetrated is very small, when compared with a knife cut, the risk of hitting an artery accidentally is lower when a needle is used. This is supported by the fact that there were no problems of bleeding or haematomas after our needle biopsies. However, colour Doppler analysis (Schlegel and Su, 1997
) was not applied.
As a procedure, needle biopsy is technically simpler and cheaper than open biopsy. It can be easily carried out in an IVF unit, which allows optimal timing as regards oocyte retrieval. Like fine needle aspiration it is much simpler than microsurgical aspiration of epididymal spermatozoa. Because it can be carried out under local anaesthesia, the patient can leave the clinic soon after the procedure.
Testicular biopty gun biopsy gives a sufficient amount of tissue for ICSI, even in cases of non-obstructive azoospermia, for histological diagnosis and for cryopreservation. The present study was retrospective, but after needle biopsy the results regarding fertilization and pregnancy rates and the proportion of men from whom spermatozoa could be obtained were at least as good as those we achieved using open surgical biopsy. We have not compared needle biopsy directly with fine needle aspiration. According to reports published earlier (Friedler et al., 1997a), fine needle aspiration does not give as good results in non-obstructive azoospermia as open biopsy. Rosenlund et al. (Rosenlund et al., 1998
) found that 21 or 19 gauge needles do not give as good histological diagnosis as open biopsies, although the method used by Craft et al. (Craft et al., 1997
) allowed histological diagnosis. Testicular needle biopsy, either by biopty gun or by butterfly needle, is the preferred methodology to open biopsy, because of its low cost, relative non-invasiveness and high success rate, with regard to the retrieval and use of testicular spermatozoa.
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Acknowledgments |
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Notes |
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References |
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Submitted on July 15, 1998; accepted on February 3, 1999.