1 ART Unit, Al Amal Hospital, PO Box 921988, Amman, Jordan and 2 Frauenklinik, Rafzeburger Allee 160-23538 Lubeck, Germany
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Abstract |
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Key words: mature spermatozoa/non-obstructive azoospermia/round spermatids/spermatid injection
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Introduction |
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Materials and methods |
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Ovarian stimulation
The protocols and procedures for female patients undergoing pituitary desensitization and follicular stimulation have been published previously, in addition to details of the ICSI procedure (Ghazzawi et al., 1998).
Spermatozoa, spermatid retrieval and processing
The injection procedure was performed on the day of egg retrieval. In patients with obstructive azoospermia, epididymal spermatozoa were retrieved either by percutaneous sperm aspiration or, when this failed, microsurgically under general anaesthesia. TESE was attempted in patients with obstructive azoospermia where epididymal sperm retrieval had failed, and in patients with non-obstructive azoospermia. The technique of TESE has been described (Ghazzawi et al., 1998). Briefly, testicular tissue was minced in HEPES-buffered Earle's medium, placed in a Falcon tube and centrifuged at 600 g throughout a discontinuous Percoll gradient in three layers (50, 70 and 90%). Spermatids were mostly found in the 50 and 70% Percoll fractions. An attempt was made to distinguish ROS from other cells such as lymphocytes, spermatocytes and Sertoli cells under an inverted (Nikon TE 300) microscope equipped with a phase-contrast system at a magnification of x200 and x400. In general, living ROS had a smooth outline, a round nucleus and a regular zone of cytoplasm surrounding them. The developing acrosomal granule could be recognized in some ROS as a bright spot adjacent to the nucleus.
Injection technique
In view of the different sizes of ROS compared with spermatozoa, we followed the technique described by Tesarik and Mendoza (1996) including modifications to the injection needle size (7 mm inner and 89 mm outer diameters), bevelled tips, and preliminary selection. This included the selection of spermatids which could stand the deformation during suction into the needle as well as regain their round shape following expulsion from the needle, and excluded those which disintegrated. Also, during injection of ROS, suction of the ooplasm was rather vigorous, as this was thought to boost oocyte activation after ICSI (Tesarik and Sousa, 1995).
Assessment of fertilization, embryo culture and transfer
Three consecutive inspections were made at 1012 h, 1618 h and 1824 h to assess fertilization. The presence of two pronuclei (2PN) was the only fertilization criterion applied. Embryonic cleavage was assessed 2 days after injection. Only embryos that had undergone at least one cleavage division were transferred to the patient's uterus.
Data analysis
Statistical analysis of discrete variables was by 2 analysis with Fisher's exact test where applicable. The differences were considered significant at P < 0.05.
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Results |
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Discussion |
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The source of ROS for microinjection is a subject of debate. Currently, it is not known what will be the best method of spermatid isolation: testicular biopsy or from the ejaculate. In a study by Fishel et al. (1997) there was a trend towards increased fertilization if ELS from testicular biopsy were utilized, but the converse was observed (i.e. round > elongated) if spermatids were isolated from the ejaculate. However, the number of cases was too small and it is too early to reach firm conclusions. On the first attempt, it is worthwhile performing extensive testicular tissue sampling in the hope of obtaining mature spermatozoa. In the event of retrieving a tiny number of spermatozoa, insufficient for injecting all oocytes, spermatid injection can give acceptable success rates (Tesarik et al., 1998). Should this attempt fail, then a search for ROS from ejaculates should be performed and would reduce irreparable testicular damage. In 87 biopsies, the majority of cells found after some hours of examination of testicular tissue were ROS, whereas Silber et al. (1996) did not find ROS in the absence of ELS. They postulated that maturation arrest could be explained by failure to progress beyond meiosis and that it is not a problem of development of the haploid cell from ROS to ELS.
Identification of ROS is a major factor which could influence the outcome of spermatid injection. In this study, we relied only on morphological criteria for identification of ROS. The observation of a round nucleus, with a regular zone of cytoplasm surrounding it, and a developing acrosome structure was more difficult with severe testicular dysfunction. Not infrequently, the nucleus was so difficult to visualize that a dark dot or slight swelling were the only useful parameters. It has been shown that injection of non-spermatid cell types or their nuclei could lead to non-specific fertilization and abnormal cleavage (Silber and Johnson, 1998; Tesarik et al., 1998
). However, confusion could be avoided by paying attention to the size of ROS, which is that of a red blood cell. There has been much effort expended in improving the recognition of spermatids in a wet preparation. After identification of a round cell based on morphological parameters under an inverted microscope (Angelopoulos et al., 1997
), or after using computer-assisted image analysis (Yamanaka et al., 1997
), the nature of such isolated cells can be verified by various techniques. Smears can be evaluated either by cytochemistry and immunocytochemistry using markers that selectively or specifically recognize the germ cell line (Mendoza and Tesarik, 1996
; Mendoza et al., 1996
), by fluorescent in-situ hybridization (FISH) to differentiate haploid spermatids from higher ploidy earlier germ cells (Mendoza et al., 1996
; Angelopoulos et al., 1997
), or by a combination of both techniques (Mendoza et al., 1996
). Since such evaluation techniques are not available in the majority of in-vitro fertilization laboratories, a simpler method was proposed by Mendoza and Tesarik (1996). They evaluated the occurrence of ROS in the ejaculates of men suffering from non-obstructive azoospermia by three different staining methods: Papanicolaou, fluorescein-labelled Pisum sativum agglutinin binding and anti-acrosin antiserum immunolabelling. Since these staining techniques are expensive and not always available, an easier method for confirming ROS selection by qualitative criteria was recently proposed by Angelopoulos et al. (1997). A rapid assessment of the morphological features of a simple cell was made after aspiration with a micropipette and staining on a prestained slide. For this method, cells <7.5 µm were selected, as larger cells are likely to be secondary spermatocytes or white blood cells.
The timing and morphology of the products of the fertilization process after ROS injection showed slight differences from that normally observed after conventional ICSI. We observed the appearance of 2PN stage in over half the zygotes as early as 1012 h after ROS injection. Previous studies have indicated that the peak time for the appearance of 2PN after ROS injection and after mature spermatozoa injection was 9 and 16 h respectively (Nagy et al., 1994; Sofikitis et al., 1995
; Yamanaka et al., 1997
). The earlier male PN formation may be due to the decondensed state of spermatids. In this study, a syngamy nucleus was observed in 36/73 initially 2PN zygotes when they were inspected again at 1618 h after ROS injection. Interestingly, the syngamy nucleus disappeared in 25 zygotes during the next inspection at 2024 h post-injection, and these zygotes cleaved the next day. This is in contrast to the findings by Tesarik and Mendoza (1996) in which all zygotes with a syngamy nucleus observed at 1618 h post-injection cleaved. It has been suggested that nuclear syngamy is a normal phenomenon in humans, but the duration of this stage depends on the male gamete maturity (short for mature spermatozoa but long for spermatids) (Tesarik and Mendoza, 1996
; Barak et al., 1998
). Although these features may be of importance for a correct evaluation of fertilization outcome after ROS injection, more research is needed to see whether such features affect further development. In this study, no pregnancies were achieved after transferring embryos developed from a syngamy nucleus, while a pregnancy followed by a live birth was achieved following transfer of embryos developing from one-nucleated zygotes after ROS injection (Barak et al., 1998
).
In the present study, the fertilization rate was significantly lower (P = 0.00001) after injection of ROS compared with injection of mature spermatozoa. This may reflect the inability of ROS originating from such severely defective testicles to achieve fertilization. The underlying causes have not yet been fully resolved; one possibility is that the cytoplasm of ROS from such pathological cases is not mature enough in comparison with its nucleus. It is possible that there is a deficiency in oocyte activating factor, with a consequential lack of Ca2+ oscillation response (Vanderzwalmen et al., 1998). Another suggestion is that the disappearance of complete spermatogenesis in all testicular tubules negatively affects the quality of spermatid maturation and may have a genetic cause (Vanderzwalmen et al., 1997
). For example, arrest of spermatogenesis can occur at the spermatid stage in cases of Y-chromosome deletions and Y-autosome translocations (Chandley, 1995
). In such a situation it is possible that the competence of ROS to fertilize and proceed to the zygote stage will be hampered. In addition, pregnancies achieved from ROS in patients with a history of spermatogenic block end in abortion, increasing the suspicion of the involvement of a genetic factor (Hannay, 1995
). Interestingly, it has been shown that spermatogenic arrest at the ROS stage can be induced experimentally in animal models (reviewed in Tesarik et al., 1998
). These observations together suggest that genetic or non-genetic abnormalities of Sertoli cells can lead to premature spermatid detachment. This is a possible pathogenetic mechanism underlying complete spermatogenesis failure in humans. The prematurely released spermatids cannot continue their differentiation due to a lack of Sertoli cell support and are finally destroyed by apoptosis (Tesarik, 1997
).
The influence of the type of immature germ cell (round, elongating, elongated spermatid) on fertilization and subsequent embryo development is a controversial issue. Although Tesarik and Mendoza (1996) reported successful pregnancies using ROS from the ejaculates, the great majority of spermatids were elongated. Vanderzwalmen et al. (1997) achieved higher fertilization after the injection of elongating and elongated spermatid. The influence of ROS injection on embryo development was evident in our results, as 38% arrested and the embryos remaining for transfer were of poor quality. In a study by Fishel et al. (1997), there was no difference in the quality of embryos resulting from ROS injection compared with conventional ICSI. In another study by Vanderzwalmen et al. (1997) the number of good quality embryos available for transfer after ROS injection was very low. Janny and Ménézo (1994) identified a strong paternal effect on embryo development and blastocyst formation. Although the hypothesis of complete embryo block could not be confirmed, is it possible that defects at DNA level in ROS prevent resultant zygotes from completing embryogenesis. In this respect, and in order to overcome the problem of developmental failure after ROS injection, Tesarik et al. (1998) suggested the application of selection methods for those few ROS which retain the relevant biological systems intact. Spermatid in-vitro culture is one possible approach which could result in replacing ROS with ELS injection, a technique which technically is much easier to perform (Tesarik and Mendoza, 1996).
In conclusion, our results reflected the reproductive capacity of ROS in patients with complete failure of spermiogenesis. Injection of ROS resulted in a significantly lower fertilization rate and a higher developmental arrest rate compared with that of mature spermatozoa. However, the observed differences might also be sue to different pathological conditions in the two groups of patients rather than to the maturation state of the germ cells alone (Vanderzwalmen et al., 1997). With no pregnancies achieved, one may question the unusual variability of the reported success rates. However, until more information about these issues is available, the technique should be applied with extreme caution after ensuring that patients are fully aware of its experimental nature.
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Notes |
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References |
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Antinori, S., Versaci, C., Dani, G. et al. (1997) Fertilization with human testicular spermatids: four successful pregnancies. Hum. Reprod., 12, 286291.[Abstract]
Araki, Y., Motoyama, M., Yoshida, A. et al. (1997) Intracytoplasmic injection with late spermatid: a successful procedure in achieving child birth for couples in which the male partner suffers from azoospermia due to deficient spermatogenesis. Fertil. Steril., 67, 559561.[ISI][Medline]
Barak, Y., Kogosowski, A., Goldman, S. et al. (1998) Pregnancy and birth after transfer of embryos that developed from single nucleated zygotes obtained by injection of round spermatids into oocytes. Fertil. Steril., 70, 6770.[ISI][Medline]
Chandley, A. (1995) The genetic basis of male infertility. Reprod. Med. Rev., 4, 18.
Devroey, P., Liu, J., Nagy, Z. et al. (1994) Normal fertilization of human oocytes after testicular sperm extraction and intracytoplasmic sperm injection (TESE and ICSI). Fertil. Steril., 62, 639641.[ISI][Medline]
Devroey, P., Liu, J., Nagy, Z. et al. (1995) Pregnancies after testicular sperm extraction and intracytoplasmic sperm injection in non-obstructive azoospermia. Hum. Reprod., 10, 14571460.[Abstract]
Edwards, R.G., Tarin, J.J., Dean, N. et al. (1994) Are spermatid injections into human oocytes now mandatory? Hum. Reprod., 9, 22172219.[ISI][Medline]
Fishel, S., Green, S., Bishop, M. et al. (1995) Pregnancy after intracytoplasmic injection of spermatid. Lancet, 345, 16411642.
Fishel, S., Aslam, I. and Tesarik, J. (1996) Spermatid conception: a stage too early, or a time too soon? Hum. Reprod., 11, 13711375.
Fishel, S., Green, S., Hunter, A. et al. (1997) Human fertilization with round and elongated spermatids. Hum. Reprod., 12, 336340.[Abstract]
Ghazzawi, I., Sarraf, M., Taher, M. and Khalifa, F. (1998) Comparison of the fertilizing capability of spermatozoa from ejaculates, epididymal aspirates and testicular biopsies using intracytoplasmic sperm injection. Hum. Reprod., 13, 348352.[Medline]
Hannay, T. (1995) New Japanese IVF method finally made available in Japan. Nature Med., 1, 289290.[ISI]
Janny, L. and Ménézo, Y. (1994) Evidence for a strong paternal effect on human preimplantation embryo development and blastocyst formation. Mol. Reprod. Dev., 38, 3642.[ISI][Medline]
Kahraman, S., Polat, G., Samli, M. et al. (1998) Multiple pregnancies obtained by testicular spermatid injection in combination with intracytoplasmic sperm injection. Hum. Reprod., 13, 104110.[Abstract]
Kimura, Y. and Yanagimachi, R. (1995) Mouse oocytes injected with testicular spermatozoa or round spermatids can develop into normal offspring. Development, 121, 23972405.
Mendoza, C. and Tesarik, J. (1996) The occurrence and identification of round spermatids in the ejaculate of men with non-obstructive azoospermia. Fertil. Steril., 66, 826829.[ISI][Medline]
Mendoza, C., Benkhalifa, M., Cohen-Bacri, P. et al. (1996) Combined use of proacrosin immunocytochemistry and autosomal DNA in situ hybridization for evaluation of human ejaculated germ cells. Zygote, 4, 279283.[ISI][Medline]
Nagy, Z.P., Liu, J. and Joris, H. (1994) Time course of oocyte activation, pronucleus formation and cleavage in human oocytes fertilized by intracytoplasmic sperm injection. Hum. Reprod., 9, 17431748.[Abstract]
Ogura, A. and Yanagimachi, R. (1993) Round spermatid nuclei injected into hamster oocytes from pronuclei and participate in syngamy. Biol. Reprod., 48, 219225.[Abstract]
Ogura, A., Yanagimachi, R. and Usui, N. (1993) Behaviour of hamster and mouse round spermatid nuclei incorporated into mature oocytes by electrofusion. Zygote, 1, 18.[Medline]
Ogura, A., Matsuda, R. and Yanagimachi, R. (1994) Birth of normal young after electrofusion of mouse oocytes with round spermatids. Proc. Natl. Acad. Sci. USA, 91, 74607462.[Abstract]
Schoysman, R., Vanderzwalmen, P., Nijs, M. et al. (1993a) Successful fertilization by testicular spermatozoa in an in vitro fertilization programme. Hum. Reprod., 8, 13391340.
Schoysman, R., Vanderzwalmen, P., Nijs, M. et al. (1993b) Pregnancy after fertilization with human testicular sperm. Lancet, 342, 1237.
Silber, S. and Johnson, L. (1998) Are spermatid injections of any clinical value. ROSNI and ROSI revisited. Hum. Reprod., 13, 509523.
Silber, S., Van Steirteghem, A., 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]
Silber, S., Van Steirteghem, A., Nagy, Z. et al. (1996) Normal pregnancies resulting from testicular sperm extraction and intracytoplasmic sperm injection from azoospermia due to maturation arrest. Fertil. Steril., 66, 110117.[ISI][Medline]
Sofikitis, N., Miyagawa, I., Agapitos, E. et al. (1994) Reproductive capacity of the nucleus of the male gamete after completion of meiosis. J. Assist. Reprod. Genet., 11, 335341.[ISI][Medline]
Sofikitis, N., Toda, T., Miyagawa, I. et al. (1995) Application of ooplasmic round spermatid nuclear injections for the treatment of azoospermic men in USA. Fertil. Steril., 64, S88S89.
Sofikitis, N., Yamamoto, Y., Miyagawa, I. et al. (1998) Ooplasmic injection of elongating spermatids for the treatment of non-obstructive azoospermia. Hum. Reprod., 13, 709714.[Abstract]
Tesarik, J. (1997) Sperm or spermatid conception. Fertil. Steril., 68, 214216.[ISI][Medline]
Tesarik, J. and Mendoza, C. (1996) Spermatid injection into human oocytes. I Laboratory techniques and special features of zygote development. Hum. Reprod., 11, 772779.[Abstract]
Tesarik, J. and Sousa, M. (1995) More than 90% fertilization rates after intracytoplasmic sperm injection and artificial induction of oocyte activation with calcium ionophore. Fertil. Steril., 63, 343349.[ISI][Medline]
Tesarik, J., Greco, E. and Mendoza, C. (1998) ROSI, instructions for use: 1997 update. Hum. Reprod., 13, 519523.[ISI][Medline]
Vanderzwalmen, P., Lejeune, B., Nijs, M. et al. (1995) Fertilization of an oocyte microinseminated with a spermatid in an in-vitro fertilization programme. Hum. Reprod., 8, 502503.
Vanderzwalmen, P., Zech, H., Birkenfeld, A. et al. (1997) Intracytoplasmic injection of spermatids retrieved from testicular tissue: influence of testicular pathology, type of selected spermatids and oocyte activation. Hum. Reprod., 12, 12031213.[ISI][Medline]
Vanderzwalmen, P., Nijs, M., Schoysman, R. et al. (1998) The problems of spermatid microinjection in the human: the need for an accurate morphological approach and selective methods for viable and normal cells. Hum. Reprod., 13, 515519.[ISI][Medline]
Yamanaka, K., Sofikitis, N. and Miyagawa, I. (1997) Ooplasmic round spermatid nuclear injection procedures as an experimental treatment for non-obstructive azoospermia. J. Assist. Reprod. Genet., 14, 5562.[ISI][Medline]
Submitted on June 22, 1998; accepted on November 10, 1998.