Assisted Reproduction Unit, American Hospital of Istanbul, Turkey
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: azoospermia/pregnancy/percutaneous testicular sperm aspiration/testicular sperm extraction
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Percutaneous epidydymal sperm aspiration (PESA) is the widely accepted and most commonly practised method of sperm retrieval when obstruction is present. In a retrospective controlled study (Tournaye et al., 1998) no difference was shown in fertilization, cleavage, and implantation rates when open biopsy was compared to fine needle aspiration in azoospermic men with normal spermatogenesis. When spermatogenesis is absent or only focal, the yield of percutaneous sperm aspiration or fine neeedle aspiration is presumed to be low and open testicular biopsy is usually preferred. However, there have been no studies evaluating the effectiveness of percutaneous testicular sperm aspiration (PTSA) in a large series of patients with non-obstructive azoospermia. In this study we report the results of 291 ICSI cycles undertaken in men with non-obstructive azoospermia where spermatozoa were searched for initially using a percutaneous approach. A TESE procedure was performed when the percutaneous approach failed to yield spermatozoa. The outcome of the two procedures was compared.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Of the 291 procedures, spermatozoa were found in 63 with PTSA (group 1), whereas a TESE had to be performed in 228 (group 2). The study period was from July 1996 to June 1999. All patients first underwent PTSA, and TESE was performed if spermatozoa were not found. Fertilization, cleavage and pregnancy rates were compared between the group who yielded spermatozoa to PTSA (group 1) and the group that proceeded to TESE (group 2).
Ovarian stimulation, oocyte retrieval and embryo transfer procedures
In the majority of treatment cycles ovarian stimulation was undertaken using subcutaneous buserelin acetate (Suprefact proinjection; Hoechst AG, Frankfurt am Main, Germany) in a long protocol combined with pure FSH (Metrodin 75; Serono, Rome, Italy). Buserelin acetate (0.3 mg/day) was commenced on day 20 or 21 of the preceding cycle and continued until the day of human chorionic gonadotrophin (HCG). In cycles where the woman was predicted to respond poorly to ovarian stimulation a flare gonadotrophin releasing hormone agonist (GnRHa) protocol was used. FSH was initiated on the third day of the menstrual cycle with 26 ampoules depending on the patient's previous or anticipated response. The treatment was then individualized in a step-down fashion. When the leading follicle reached 20 mm in mean diameter with a serum oestradiol concentration of 200300 pg/ml per mature follicle 10 000 U HCG (Profasi HP 5000; Serono) was administered. Oocyte retrieval was performed 36 h after the injection of HCG. ICSI was performed according to conventional protocols (Van Steirteghem et al., 1993) and only on metaphase II oocytes. Two to five embryos were replaced 2 or 3 days after retrieval according to number of embryos available, embryo quality and age of the woman. A serum pregnancy test was performed 1012 days after embryo transfer. Clinical pregnancy was defined as the presence of gestational sac/s with a viable embryo shown on vaginal ultrasonography performed ~24 days after embryo transfer.
Testicular sperm aspiration and extraction procedures
Both PTSA and TESE specimens were processed in the same manner. Procedures were performed under general anaesthesia or local anaesthesia with i.v. sedation. All biopsies were performed bilaterally if spermatozoa were not retrieved from the first side. All procedures were performed 2448 h prior to oocyte retrieval. This eased the workload on laboratory and operating room personnel. Results from a previous study showed that this had no deleterious effect on sperm viability (Urman et al., 1998). Scheduling PTSA/TESE 48 h prior to oocyte retrieval also obviated the need to administer HCG to the woman at risk of developing ovarian hyperstimulation syndrome in case spermatozoa could not be retrieved. PTSA was performed with a 21-gauge Butterfly needle that was inserted into the testes and moved up and down to sample a wide area. An artery forceps was secured across the attached microtubing set before the needle was withdrawn. The aspirate located in the tubing was washed into a Falcon tube (Becton Dickinson, Franklin Lakes, NJ, USA) with a small volume of media. The presence of spermatozoa was sought under x200 magnification. Percutaneous aspiration was attempted from three different areas of the testis and if spermatozoa were not observed a TESE procedure was performed.
Tissue samples measuring in size from 0.5x0.5x0.5 to 1x1x1 cm were removed until spermatozoa were identified or 45 biopsy pieces were extracted from each testis. Testicular tissue samples thus obtained were placed into Falcon tubes containing 2 ml EBSS medium (Earle's balanced salt solution; Sigma, Aldrich Co. Ltd, Irvine, UK) supplemented with HSA (human serum albumin; Irvine Scientific, Santa Ana, CA, USA) penicillin and pyruvate. Tissue samples were then divided into small segments, and gently crushed with micro-needles in a Petri dish containing the same medium. The sperm suspension thus obtained was then centrifuged for 5 min at 300 g in a Falcon tube. If sperm cells were identified, the suspension was centrifuged on a two-layer Percoll gradient (50% and 90%) to free the sperm from debris and red blood cells. Both gradients were washed with HSA-supplemented EBSS. Following centrifugation, the supernatant was removed and the final pellet in the HAS-supplemented EBSS solution was prepared as a swim-out droplet covered with mineral oil (Sigma). The droplet was incubated at 37°C in 5% CO2 until the ICSI procedure. As part of a concurrent study, in-vitro culture of testicular tissue samples with recombinant FSH was undertaken in 143 cycles (Balaban et al., 1999).
Statistical analysis
The age of patients, duration of stimulation, number of ampoules used, number of embryos transferred, oestradiol on the day of HCG, number of eggs retrieved and endometrial thickness were compared using t-test; pregnancy rates were compared using 2-test. P < 0.05 was accepted as significant.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It has been shown that abnormal sperm decondensation is an important component of fertilization failure (Dozortsev et al., 1994). Alterations in sperm chromatin might result in defective decondensation and DNA activation during fertilization that may result in fertilization failure, early embryonic wastage or poor embryonic development (Hamamah et al., 1997
). It is also known that maternal genes control the first two cell division cycles of the embryo (Braude et al., 1988
). The paternal genetic effect is not exerted until the 4-cell stage; therefore, any potential detrimental paternal effect would only be observed after that particular stage. Evidence has been provided for a strong paternal effect on human preimplantation embryo development by studying blastocyst formation in co-culture (Janny and Ménézo, 1994
). These workers compared blastocyst formation in IVF cycles undertaken with either normal spermatozoa, donor spermatozoa, or spermatozoa from severely oligozoospermic men. The overall number of blastocysts and the number of patients having at least one blastocyst were severely reduced when spermatozoa from severely oligozoospermic men were used to inseminate the oocytes.
Delayed fertilization and poor embryonic development were reported to be associated with impaired semen quality following IVF (Ron-El et al., 1991). It was later suggested that the spermatozoon is involved in the embryonic quality, even in early stages of development, and that this shows an association between abnormal sperm morphology and poor embryo morphology (Parinaud et al., 1993
).
It is possible that the severity of spermatogenic defect is associated with ease of sperm retrieval and affects embryo implantation. There may be chromatin defects and DNA abnormalities in spermatozoa from men with non-obstructive azoospermia and the frequency of these abnormalities may be related to testicular pathology or the severity of spermatogenic impairment. It seems reasonable to conclude from our results that, in couples with non-obstructive azoospermia, implantation potential is significantly lower in patients who require TESE after failing PTSA. The difference is most probably due to the severity of testicular pathology, i.e. the more severe the spermatogenic defect, the more difficult the retrieval of spermatozoa from the testis.
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Balaban, B., Urman, B., Sertac, A. et al. (1999) In-vitro culture of spermatozoa induces motility and increases implantation and pregnancy rates after testicular sperm extraction and intracytoplasmic sperm injection. Hum. Reprod., 14, 28082811.
Bernardini, L., Borini, A., Preti, S. et al. (1998) Study of aneuploidy in normal and abnormal germ cells from semen of fertile and infertile men. Hum. Reprod., 13, 34063413.[Abstract]
Braude, P., Bolton, V. and Moore, S. (1988) Human gene expression first occurs between the four- and eight-cell stages of preimplantation development. Nature, 332, 459461.[ISI][Medline]
Chandley, A.C. (1995) The genetic basis of male infertility. Reprod. Med. Rev., 4, 18.
Craft, I., Bennett, V. and Nicholsson, N. (1993) Fertilizing ability of testicular spermatozoa. Lancet, 349, 864.
Dozortsev, D., De Sutter, P. and Dhont, M. (1994) Behaviour of spermatozoa in human oocytes displaying no or one pronucleus after intracytoplasmic sperm injection. Hum. Reprod., 9, 21392144.[Abstract]
Fahmy, I., Mansour, R., Aboulghar, M. et al. (1996) Intracytoplasmic sperm injection of surgically retrieved sperm and spermatids in patients with obstructive and nonobstructive azoospermia. Middle East Fertil. Soc.. J., 1, 134141.
Ghazzawi, I.M., Sarraf, M.G., Taher, M.R. and Khalifa, F.A. (1998) Comparison of fertilizing capability of spermatozoa from ejaculates, epididymal aspirates and testicular biopsies using intracytoplasmic sperm injection. Hum. Reprod., 13, 34852.[Medline]
Hamamah, S., Fignon, A. and Lansac, J. (1997) The effect of male factors in repeated spontaneous abortion: lesson from in vitro fertilization and intracytoplasmic sperm injection. Hum. Reprod. Update, 3, 393400.
In't Veld, P.A., Broekman, F.J.M., de France, H.F. et al. (1997) Intracytoplasmic sperm injection (ICSI) and chromosomally abnormal spermatozoa. Hum. Reprod., 12, 752754.[Abstract]
Rejio, R., Lee, T., Salo, P. et al. (1995) Diverse spermatogenetic defects in humans caused by Y chromosome deletions encompassing a novel RNA-binding gene. Nat. Genet., 10, 383393.[ISI][Medline]
Janny, L. and Ménézo, Y.J.R. (1994) Evidence for a strong paternal effect on human preimplantation embryo development and blastocyst formation. Mol. Reprod. Dev., 38, 3642.[ISI][Medline]
Johannisson, R., Schwinger E., Wolff, H.H. et al. (1993) The effect of 13;14 Robertsonian translocation on germ cell differentation in infertile males. Cytogenet. Cell. Genet., 63, 151155.[ISI][Medline]
Kahraman, S., Ozgur, S., Alatas, C. et al. (1996) High implantation and pregnancy rates with testicular sperm extraction and intracytoplasmic sperm injection in obstructive and non-obstructive azoospermia. Hum. Reprod., 11, 673676.[Abstract]
Koyabashi, K., Mizuno, K., Hida, A. et al. (1994) PCR analysis of the Y chromosome long arm in azoospermic patients: evidence for second locus required for spermatogenesis. Hum. Mol. Genet., 3, 19651967.[Abstract]
Lahdeite, J., Saari, N., Ajosenpaa-Saari, M. and Mykkanen, J. (1997) Incidence of aneuoploid spermatozoa among infertile men studied by multicolor fluorescence in-situ hybridization. Am. J. Med. Genet., 71, 115121.[ISI][Medline]
Ma, K., Sharkey, A., Kirsch, S. et al. (1992) Towards the molecular localization of the AZF locus: mapping of microdeletions in azoospermic men within 14 subintervals of interval 6 of the human Y chromosome. Hum. Mol. Genet., 1, 2933.[Abstract]
Mansour, R.T., Kamal, A., Fahmy, I. et al. (1997) Intracytoplasmic sperm injection in obstructive and nonobstructive azoospermia. Hum. Reprod., 12, 19741979.[Abstract]
Martin, R., Spriggs, E. and Rademaker, A.W. (1996) Multicolor fluorescence in-situ hybridization analysis of aneuploidy and diploidy frequencies in 225846 sperm from 10 normal men. Biol. Reprod., 54, 394398.[Abstract]
Moosani, N., Pattinson, H.A., Carter. M.D. et al. (1995) Chromosomal analysis of sperm from men with idiopathic infertility using sperm karyotyping and fluorescence in-situ hybridization. Fertil. Steril., 64, 811817.[ISI][Medline]
Pang, M.G., Zackowski, J.L., Hoegerman, S.F. et al. (1995) Detection by fluorescence in situ hybridization of chromosome 7, 11, 12, 18, X and Y abnormalities in sperm from oligoasthenospermic patients of an in vitro fertilization program. J. Assist. Reprod. Genet., 12 (Suppl.), 53S.
Parinaud, J., Mieusset, R, Vieitez, G. et al. (1993) Influence of sperm parameters on embryo quality. Fertil. Steril., 60, 888892.[ISI][Medline]
Retief, A.E., van Zyl, J.A., Menkveld, M.F. et al. (1984) Chromosome studies in 496 infertile males with a sperm counts below 10 million per ml. Hum. Genet., 66, 162164.[ISI][Medline]
Ron-El, R., Nachum, H., Herman, A. et al. (1991) Delayed fertilization and poor embryonic development associated with impaired semen quality. Fertil. Steril., 55, 338344.[ISI][Medline]
Silber, S.J., Van Steirteghem, A.C., Liu, J. et al. (1995) High fertilization and pregnancy rate after intracytoplasmic sperm injection with spermatozoa obtained from testicle biopsy. Hum. Reprod., 10, 148152.[Abstract]
Staessen, C., Camus, M., Bollen, N. et al. (1992) The relationship between embyo quality and the occurrence of multiple pregnancies. Fertil. Steril., 3, 626630.
Tarlatzis, B. (1996) Report on the activities of the ESHRE Task Force on intracytoplasmic sperm injection. In Van Steirteghem, A., Devroey, P. and Liebaers, (eds), Genetics and Assisted Human Conception. Oxford University Press, Oxford, pp. 160176.
Tournaye, H., Clasen, K., Aytoz, A. et al. (1998) Fine needle aspiration versus open biopsy for testicular sperm recovery: a controlled study in azoospermic patients with normal spermatogenesis. Hum. Reprod., 13, 901904.[Abstract]
Urman, B., Alatas, C., Aksoy, S. et al. (1998) Performing testicular or epididymal sperm retrieval prior to the injection of hCG. J. Assist. Reprod. Genet., 15, 125128.[ISI][Medline]
Van Steirteghem, A., Nagy, Z., Joris, H. et al. (1993) High fertilization and implantation rates after intracytoplasmic sperm injection. Hum. Reprod., 8, 10611066.[Abstract]
Yoshida, A., Tamayama, T. and Nagao, K. (1995) A cytogenetic survey of 1007 infertile males. Contracept. Fertil. Sex., 23 (Suppl. 9), S23.
Submitted on December 30, 1999; accepted on April 3, 2000.