Round spermatid nucleus injection (ROSNI)

Abstract

BackgroundROSNI, or ROSI (round spermatid injection) is a method of assisted in vitro fertilization (IVF) in which precursors of mature sperm obtained by ejaculated specimens or testicular sperm extraction (TESE) are injected directly into oocytes as treatment for male infertility. ROSNI has been proposed as a treatment for men in whom mature sperm forms (elongating spermatids or spermatozoa) cannot be identified for intracytoplasmic sperm injection (ICSI), as in men with complete meiotic arrest. ROSNI has not been widely performed or as successful as ICSI, which revolutionized male infertility.Because ROSNI involves the use of immature sperm, the procedure presents new technical challenges and raises new unresolved genetic concerns. The Practice Committee recommends that ROSNI be considered an experimental procedure that should be applied only in the setting of a clinical trial approved and overseen by a properly constituted Institutional Review Board.SpermatogenesisSpermatogenesis can be divided into three major stages. The first stage entails mitotic division of the spermatogonia to yield diploid (2N) primary spermatocytes. In the second stage, primary spermatocytes undergo two successive meiotic divisions. The first gives rise to haploid (1N) secondary spermatocytes, and the second to round spermatids. In the final stage of spermatogenesis (termed spermiogenesis), round spermatids further mature, first becoming elongated spermatids (lacking a defined tail) and, finally, mature spermatozoa.It has been proposed that differentiation of spermatids into mature spermatozoa serves only to provide the means to transport paternal genetic information to the oocyte. Therefore, in theory, the injected haploid genome of the round spermatid nucleus may be sufficient for fertilization and subsequent embryonic development in some species. In the mouse model, injection of round spermatids (1Ogura A. Matsuda J. Asano T. Suzuki O. Yanagimachi R. Mouse oocytes injected with cryopreserved round spermatids can develop into normal offspring.J Assist Reprod Genet. 1996; 13: 431-434Crossref PubMed Scopus (65) Google Scholar, 2Ogura A. Matsuda J. Yanagimachi R. Birth of normal young after electrofusion of mouse oocytes with round spermatids.Proc Natl Acad Sci USA. 1994; 91: 7460-7462Crossref PubMed Scopus (258) Google Scholar, 3Ogura A. Yanagimachi R. Round spermatid nuclei injected into hamster oocytes from pronuclei and participate in syngamy.Biol Reprod. 1993; 48: 219-225Crossref PubMed Scopus (160) Google Scholar, 4Kimura Y. Yanagimachi R. Mouse oocytes injected with testicular spermatozoa or round spermatids can develop into normal offspring.Development. 1995; 121: 2397-2405PubMed Google Scholar) and secondary spermatocytes (5Kimura Y. Yanagimachi R. Development of normal mice from oocytes injected with secondary spermatocyte nuclei.Biol Reprod. 1995; 53: 855-862Crossref PubMed Scopus (198) Google Scholar) into oocytes has achieved fertilization and pregnancy with some consistency (6Tamashiro K.L. Kimura Y. Blanchard R.J. Blanchard D.C. Yanagimachi R. Bypassing spermiogenesis for several generations does not have detrimental consequences on the fertility and neurobehavior of offspring a study using the mouse.J Assist Reprod Genet. 1999; 16: 315-324Crossref PubMed Scopus (23) Google Scholar). However, in at least one strain of sterile male mice tested with ROSNI, the phenotypic abnormality causing sterility in the father was more severely expressed in the offspring, results that illustrate one potential risk of this technology (7Akutsu H. Tres L.L. Tateno H. Yanagimachi R. Kierszenbaum A.L. Off-spring from normal mouse oocytes injected with sperm heads from the azh/azh mouse display more severe sperm tail abnormalities than the original mutant.Biol Reprod. 2001; 64: 249-256Crossref PubMed Scopus (35) Google Scholar).Unresolved issues associated with rosniRound spermatid identificationROSNI has been used to achieve fertilization in human IVF, but with limited success (8Gianaroli L. Selman H.A. Magli M.C. Colpi G. Fortini D. Ferraretti A.P. Birth of a healthy infant after conception with round spermatids isolated from cryopreserved testicular tissue.Fertil Steril. 1999; 72: 539-541Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 9Tesarik J. Cruz-Navarro N. Moreno E. Canete M.T. Mendoza C. Birth of healthy twins after fertilization with in vitro cultured spermatids from a patient with massive in vivo apoptosis of postmeiotic germ cells.Fertil Steril. 2000; 74: 1044-1046Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 10Schoysman R. Vanderzwalmen P. Bertin G. Nijs M. Van Damme B. Oocyte insemination with spermatozoa precursors.Curr Opin Urol. 1999; 9: 541-545Crossref PubMed Scopus (12) Google Scholar). Accurate identification of round spermatid cells is one technical challenge of ROSNI. Using the standard optics present in most clinical IVF laboratories, it can be difficult to distinguish haploid round spermatid cells from diploid spermatogenic precursors and somatic cells. Phase contrast microscopy may simplify the task (11Verheyen G. Crabbe E. Joris H. Van Steirteghem A. Simple and reliable identification of the human round spermatid by inverted phase-contrast microscopy.Hum Reprod. 1998; 13: 1570-1577Crossref PubMed Scopus (34) Google Scholar). Another technique recently described uses a fluorescent mitochondrial probe to specifically identify round spermatids in dispersed testicular cells (12Sutovsky P. Ramalho-Santos J. Moreno R.D. Oko R. Hewitson L. Schatten G. On-stage selection of single round spermatids using a vital, mitochondrion-specific fluorescent probe MitoTracker(TM) and high resolution differential interference contrast microscopy.Hum Reprod. 1999; 14: 2301-2312Crossref PubMed Scopus (43) Google Scholar). Originally developed for use in nonhuman primate species and cattle, the technique will require extensive testing to establish its safety before it can be applied in humans.Oocyte activationActivation of the oocyte rarely occurs after spermatid injection alone. This observation suggests that another factor present in mature spermatozoa may serve to induce a calcium flux in the oocyte, resulting in its activation (13Tesarik J. Mendoza C. Greco E. The activity (calcium oscillator?) responsible for human oocyte activation after injection with round spermatids is associated with spermatid nuclei.Fertil Steril. 2000; 74: 1245-1247Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 14Yazawa H. Yanagida K. Katayose H. Hayashi S. Sato A. Comparison of oocyte activation and Ca2+ oscillation-inducing abilities of round/elongated spermatids of mouse, hamster, rat, rabbit and human assessed by mouse oocyte activation assay.Hum Reprod. 2000; 15: 2582-2590Crossref PubMed Scopus (51) Google Scholar, 15Yanagida K. Yazawa H. Katayose H. Kimura Y. Hayashi S. Sato A. Oocyte activation induced by spermatids and the spermatozoa.Int J Androl. 2000; 23: 63-65Crossref PubMed Scopus (17) Google Scholar). Extraction and delivery of this factor or treatment with a calcium ionophore are two potential strategies for inducing oocyte activation, which is required for fertilization.Embryonic developmentIn primate species, the embryonic centrosome normally derives from spindle elements contributed by spermatozoa. Because round spermatids have not yet developed mature centriolar complexes with spindles, their injection into oocytes has the theoretical potential for unexpected and possibly adverse effects on subsequent embryonic development.Genetic abnormalitiesOur current understanding of the molecular control mechanisms that regulate spermatogenesis, sperm transport, fertilization, and early embryonic development is severely limited. Consequently, nearly one quarter of all male infertility is categorized as idiopathic. Among men with nonobstructive azoospermia who are candidates for TESE, only approximately 17% have identifiable genetic abnormalities. Most affected men have chromosomal anomalies, predominantly Klinefelter syndrome (46,XXY); 7% to 10% harbor known Y chromosome microdeletions (16Rucker G.B. Mielnik A. King P. Goldstein M. Schlegel P.N. Preoperative screening for genetic abnormalities in men with nonobstructive azoospermia before testicular sperm extraction.J Urol. 1998; 160: 2068-2071Abstract Full Text Full Text PDF PubMed Google Scholar).Among remaining azoospermic men, some are likely to have as yet unidentified autosomal genetic defects (17Lamb D.J. Debate is ICSI a genetic time bomb? Yes.J Androl. 1999; 20: 23-33PubMed Google Scholar). Certainly, any genetic abnormality sufficiently severe to result in meiotic arrest (the only cause of infertility that could possibly benefit from application of ROSNI) might also have adverse effects on other normal cellular processes or other systemic manifestations. Offspring conceived via IVF with ROSNI may thus be at risk for infertility or even more severe genetic defects.The health consequences for offspring conceived through ROSNI are largely unknown. An early report suggested that IVF with ICSI might be associated with an increased risk for sex chromosome abnormalities (18Bonduelle M. Aytoz A. Van Assche E. Devroey P. Liebaers I. Van Steirteghem A. Incidence of chromosomal aberrations in children born after assisted reproduction through intracytoplasmic sperm injection.Hum Reprod. 1998; 13: 781-782Crossref PubMed Scopus (184) Google Scholar). Subsequent studies have suggested that ICSI has no intrinsic genetic risks (19Bonduelle M. Camus M. De Vos A. Staessen C. Tournaye H. Van Assche E. et al.Seven years of intracytoplasmic sperm injection and follow-up of 1987 subsequent children.Hum Reprod. 1999; 14: 243-264Crossref PubMed Scopus (216) Google Scholar, 20Bonduelle M. Wilikens A. Buysse A. et al.A follow-up study of children born after intracytoplasmic sperm injection (ICSI) with epididymal and testicular spermatozoa and after replacement of cryopreserved embryos obtained after ICSI.Hum Reprod. 1998; 13: 196-207Crossref PubMed Scopus (91) Google Scholar, 21Tarlatzis B.C. Report on the activities of the ESHRE Task Force on intracytoplasmic sperm injection. European Society of Human Reproduction and Embryology.Hum Reprod. 1996; 11: 160-185Crossref PubMed Scopus (29) Google Scholar) or that risk of birth defects is increased with IVF, but to no greater extent when ICSI is also performed using ejaculated sperm (22Hansen M. Kurinczuk J.J. Bower C. Webb S. The risk of major birth defects after intracytoplasmic sperm injection and in vitro fertilization.N Engl J Med. 2002; 346: 725-730Crossref PubMed Scopus (917) Google Scholar). However, data regarding the safety of ICSI using testicular sperm or spermatids obtained from men with nonobstructive azoospermia are far more limited.Genomic imprinting (modification of gene expression by cytosine methylation) normally occurs during gametogenesis and may not be complete early in the round spermatid stage of development. Naturally occurring errors in imprinting may result in illness, such as type II Angelman syndrome (a genetic disorder characterized by mental retardation, lack of speech, and movement and behavior disorders). In mice, genomic imprinting is completed in the round spermatid (23Shamanski F.L. Kimura Y. Lavoir M.C. Pedersen R.A. Yanagimachi R. Status of genomic imprinting in mouse spermatids.Hum Reprod. 1999; 14: 1050-1056Crossref PubMed Scopus (78) Google Scholar), but the exact stage of genomic imprinting in human spermatogenesis is unknown and must be defined before clinical application of ROSNI in human IVF.Limited success ratesTo date, the application of ROSNI in clinical IVF has had disappointing results. Although several reports have suggested the feasibility of ROSNI in human IVF (8Gianaroli L. Selman H.A. Magli M.C. Colpi G. Fortini D. Ferraretti A.P. Birth of a healthy infant after conception with round spermatids isolated from cryopreserved testicular tissue.Fertil Steril. 1999; 72: 539-541Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 9Tesarik J. Cruz-Navarro N. Moreno E. Canete M.T. Mendoza C. Birth of healthy twins after fertilization with in vitro cultured spermatids from a patient with massive in vivo apoptosis of postmeiotic germ cells.Fertil Steril. 2000; 74: 1044-1046Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 10Schoysman R. Vanderzwalmen P. Bertin G. Nijs M. Van Damme B. Oocyte insemination with spermatozoa precursors.Curr Opin Urol. 1999; 9: 541-545Crossref PubMed Scopus (12) Google Scholar, 24Al-Hasani S. Ludwig M. Palermo I. et al.Intracytoplasmic injection of round and elongated spermatids from azoospermic patients results and review.Hum Reprod. 1999; 14: 97-107Crossref PubMed Scopus (35) Google Scholar, 25Choavaratana R. Suppinyopong S. Chaimahaphruksa P. ROSI from TESE the first case in Thailand a case report.J Med Assoc Thai. 1999; 82: 938-941PubMed Google Scholar), overall fertilization rates achieved with ROSNI are lower (45% to 50%) than with ICSI using mature sperm or elongating spermatids (69% to 74%) (10Schoysman R. Vanderzwalmen P. Bertin G. Nijs M. Van Damme B. Oocyte insemination with spermatozoa precursors.Curr Opin Urol. 1999; 9: 541-545Crossref PubMed Scopus (12) Google Scholar, 26Vicdan K. Isik A.Z. Delilbasi L. Development of blastocyst-stage embryos after round spermatid injection in patients with complete spermiogenesis failure.J Assist Reprod Genet. 2001; 18: 78-86Crossref PubMed Scopus (29) Google Scholar, 27Levran D. Nahum H. Farhi J. Weissman A. Poor outcome with round spermatid injection in azoospermic patients with maturation arrest.Fertil Steril. 2000; 74: 443-449Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 28Balaban B. Urman B. Isiklar A. Alatas C. Aksoy S. Mercan R. et al.Progression to the blastocyst stage of embryos derived from testicular round spermatids.Hum Reprod. 2000; 15: 1377-1382Crossref PubMed Scopus (44) Google Scholar). One report on the occurrence of significant congenital anomalies in ROSNI-conceived pregnancies raises further concerns (29Zech H. Vanderzwalmen P. Prapas Y. Lejeune B. Duba E. Schoysman R. Congenital malformations after intracytoplasmic injection of spermatids.Hum Reprod. 2000; 15: 969-971Crossref PubMed Scopus (58) Google Scholar). Although causality was not established, the report indicates that caution is warranted.Summary and recommendations·ROSNI is a method of assisted fertilization in which precursors of mature spermatozoa are injected into oocytes.·Accurate identification of round spermatids remains a technical challenge.·Other important unresolved issues include impaired oocyte activation, potential adverse effects on the embryonic centrosome, and risk of infertility or more severe genetic defects in offspring.·The health consequences of ROSNI for offspring are uncertain.·Further research is required to better define the genetic causes of male infertility and to establish the safety of ROSNI and other novel assisted reproductive technologies.·ROSNI should not be performed when more mature sperm forms (elongating spermatids or spermatozoa) can be identified and used for ICSI.·Patients who may be candidates for ROSNI should receive careful and thorough pretreatment counseling to ensure they are clearly informed of the limitations and potential risks of the procedure.·Application of ROSNI in clinical human IVF should be considered experimental and therefore requires approval and oversight by an appropriately constituted Institutional Review Board. BackgroundROSNI, or ROSI (round spermatid injection) is a method of assisted in vitro fertilization (IVF) in which precursors of mature sperm obtained by ejaculated specimens or testicular sperm extraction (TESE) are injected directly into oocytes as treatment for male infertility. ROSNI has been proposed as a treatment for men in whom mature sperm forms (elongating spermatids or spermatozoa) cannot be identified for intracytoplasmic sperm injection (ICSI), as in men with complete meiotic arrest. ROSNI has not been widely performed or as successful as ICSI, which revolutionized male infertility.Because ROSNI involves the use of immature sperm, the procedure presents new technical challenges and raises new unresolved genetic concerns. The Practice Committee recommends that ROSNI be considered an experimental procedure that should be applied only in the setting of a clinical trial approved and overseen by a properly constituted Institutional Review Board. ROSNI, or ROSI (round spermatid injection) is a method of assisted in vitro fertilization (IVF) in which precursors of mature sperm obtained by ejaculated specimens or testicular sperm extraction (TESE) are injected directly into oocytes as treatment for male infertility. ROSNI has been proposed as a treatment for men in whom mature sperm forms (elongating spermatids or spermatozoa) cannot be identified for intracytoplasmic sperm injection (ICSI), as in men with complete meiotic arrest. ROSNI has not been widely performed or as successful as ICSI, which revolutionized male infertility. Because ROSNI involves the use of immature sperm, the procedure presents new technical challenges and raises new unresolved genetic concerns. The Practice Committee recommends that ROSNI be considered an experimental procedure that should be applied only in the setting of a clinical trial approved and overseen by a properly constituted Institutional Review Board. SpermatogenesisSpermatogenesis can be divided into three major stages. The first stage entails mitotic division of the spermatogonia to yield diploid (2N) primary spermatocytes. In the second stage, primary spermatocytes undergo two successive meiotic divisions. The first gives rise to haploid (1N) secondary spermatocytes, and the second to round spermatids. In the final stage of spermatogenesis (termed spermiogenesis), round spermatids further mature, first becoming elongated spermatids (lacking a defined tail) and, finally, mature spermatozoa.It has been proposed that differentiation of spermatids into mature spermatozoa serves only to provide the means to transport paternal genetic information to the oocyte. Therefore, in theory, the injected haploid genome of the round spermatid nucleus may be sufficient for fertilization and subsequent embryonic development in some species. In the mouse model, injection of round spermatids (1Ogura A. Matsuda J. Asano T. Suzuki O. Yanagimachi R. Mouse oocytes injected with cryopreserved round spermatids can develop into normal offspring.J Assist Reprod Genet. 1996; 13: 431-434Crossref PubMed Scopus (65) Google Scholar, 2Ogura A. Matsuda J. Yanagimachi R. Birth of normal young after electrofusion of mouse oocytes with round spermatids.Proc Natl Acad Sci USA. 1994; 91: 7460-7462Crossref PubMed Scopus (258) Google Scholar, 3Ogura A. Yanagimachi R. Round spermatid nuclei injected into hamster oocytes from pronuclei and participate in syngamy.Biol Reprod. 1993; 48: 219-225Crossref PubMed Scopus (160) Google Scholar, 4Kimura Y. Yanagimachi R. Mouse oocytes injected with testicular spermatozoa or round spermatids can develop into normal offspring.Development. 1995; 121: 2397-2405PubMed Google Scholar) and secondary spermatocytes (5Kimura Y. Yanagimachi R. Development of normal mice from oocytes injected with secondary spermatocyte nuclei.Biol Reprod. 1995; 53: 855-862Crossref PubMed Scopus (198) Google Scholar) into oocytes has achieved fertilization and pregnancy with some consistency (6Tamashiro K.L. Kimura Y. Blanchard R.J. Blanchard D.C. Yanagimachi R. Bypassing spermiogenesis for several generations does not have detrimental consequences on the fertility and neurobehavior of offspring a study using the mouse.J Assist Reprod Genet. 1999; 16: 315-324Crossref PubMed Scopus (23) Google Scholar). However, in at least one strain of sterile male mice tested with ROSNI, the phenotypic abnormality causing sterility in the father was more severely expressed in the offspring, results that illustrate one potential risk of this technology (7Akutsu H. Tres L.L. Tateno H. Yanagimachi R. Kierszenbaum A.L. Off-spring from normal mouse oocytes injected with sperm heads from the azh/azh mouse display more severe sperm tail abnormalities than the original mutant.Biol Reprod. 2001; 64: 249-256Crossref PubMed Scopus (35) Google Scholar). Spermatogenesis can be divided into three major stages. The first stage entails mitotic division of the spermatogonia to yield diploid (2N) primary spermatocytes. In the second stage, primary spermatocytes undergo two successive meiotic divisions. The first gives rise to haploid (1N) secondary spermatocytes, and the second to round spermatids. In the final stage of spermatogenesis (termed spermiogenesis), round spermatids further mature, first becoming elongated spermatids (lacking a defined tail) and, finally, mature spermatozoa. It has been proposed that differentiation of spermatids into mature spermatozoa serves only to provide the means to transport paternal genetic information to the oocyte. Therefore, in theory, the injected haploid genome of the round spermatid nucleus may be sufficient for fertilization and subsequent embryonic development in some species. In the mouse model, injection of round spermatids (1Ogura A. Matsuda J. Asano T. Suzuki O. Yanagimachi R. Mouse oocytes injected with cryopreserved round spermatids can develop into normal offspring.J Assist Reprod Genet. 1996; 13: 431-434Crossref PubMed Scopus (65) Google Scholar, 2Ogura A. Matsuda J. Yanagimachi R. Birth of normal young after electrofusion of mouse oocytes with round spermatids.Proc Natl Acad Sci USA. 1994; 91: 7460-7462Crossref PubMed Scopus (258) Google Scholar, 3Ogura A. Yanagimachi R. Round spermatid nuclei injected into hamster oocytes from pronuclei and participate in syngamy.Biol Reprod. 1993; 48: 219-225Crossref PubMed Scopus (160) Google Scholar, 4Kimura Y. Yanagimachi R. Mouse oocytes injected with testicular spermatozoa or round spermatids can develop into normal offspring.Development. 1995; 121: 2397-2405PubMed Google Scholar) and secondary spermatocytes (5Kimura Y. Yanagimachi R. Development of normal mice from oocytes injected with secondary spermatocyte nuclei.Biol Reprod. 1995; 53: 855-862Crossref PubMed Scopus (198) Google Scholar) into oocytes has achieved fertilization and pregnancy with some consistency (6Tamashiro K.L. Kimura Y. Blanchard R.J. Blanchard D.C. Yanagimachi R. Bypassing spermiogenesis for several generations does not have detrimental consequences on the fertility and neurobehavior of offspring a study using the mouse.J Assist Reprod Genet. 1999; 16: 315-324Crossref PubMed Scopus (23) Google Scholar). However, in at least one strain of sterile male mice tested with ROSNI, the phenotypic abnormality causing sterility in the father was more severely expressed in the offspring, results that illustrate one potential risk of this technology (7Akutsu H. Tres L.L. Tateno H. Yanagimachi R. Kierszenbaum A.L. Off-spring from normal mouse oocytes injected with sperm heads from the azh/azh mouse display more severe sperm tail abnormalities than the original mutant.Biol Reprod. 2001; 64: 249-256Crossref PubMed Scopus (35) Google Scholar). Unresolved issues associated with rosniRound spermatid identificationROSNI has been used to achieve fertilization in human IVF, but with limited success (8Gianaroli L. Selman H.A. Magli M.C. Colpi G. Fortini D. Ferraretti A.P. Birth of a healthy infant after conception with round spermatids isolated from cryopreserved testicular tissue.Fertil Steril. 1999; 72: 539-541Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 9Tesarik J. Cruz-Navarro N. Moreno E. Canete M.T. Mendoza C. Birth of healthy twins after fertilization with in vitro cultured spermatids from a patient with massive in vivo apoptosis of postmeiotic germ cells.Fertil Steril. 2000; 74: 1044-1046Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 10Schoysman R. Vanderzwalmen P. Bertin G. Nijs M. Van Damme B. Oocyte insemination with spermatozoa precursors.Curr Opin Urol. 1999; 9: 541-545Crossref PubMed Scopus (12) Google Scholar). Accurate identification of round spermatid cells is one technical challenge of ROSNI. Using the standard optics present in most clinical IVF laboratories, it can be difficult to distinguish haploid round spermatid cells from diploid spermatogenic precursors and somatic cells. Phase contrast microscopy may simplify the task (11Verheyen G. Crabbe E. Joris H. Van Steirteghem A. Simple and reliable identification of the human round spermatid by inverted phase-contrast microscopy.Hum Reprod. 1998; 13: 1570-1577Crossref PubMed Scopus (34) Google Scholar). Another technique recently described uses a fluorescent mitochondrial probe to specifically identify round spermatids in dispersed testicular cells (12Sutovsky P. Ramalho-Santos J. Moreno R.D. Oko R. Hewitson L. Schatten G. On-stage selection of single round spermatids using a vital, mitochondrion-specific fluorescent probe MitoTracker(TM) and high resolution differential interference contrast microscopy.Hum Reprod. 1999; 14: 2301-2312Crossref PubMed Scopus (43) Google Scholar). Originally developed for use in nonhuman primate species and cattle, the technique will require extensive testing to establish its safety before it can be applied in humans.Oocyte activationActivation of the oocyte rarely occurs after spermatid injection alone. This observation suggests that another factor present in mature spermatozoa may serve to induce a calcium flux in the oocyte, resulting in its activation (13Tesarik J. Mendoza C. Greco E. The activity (calcium oscillator?) responsible for human oocyte activation after injection with round spermatids is associated with spermatid nuclei.Fertil Steril. 2000; 74: 1245-1247Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 14Yazawa H. Yanagida K. Katayose H. Hayashi S. Sato A. Comparison of oocyte activation and Ca2+ oscillation-inducing abilities of round/elongated spermatids of mouse, hamster, rat, rabbit and human assessed by mouse oocyte activation assay.Hum Reprod. 2000; 15: 2582-2590Crossref PubMed Scopus (51) Google Scholar, 15Yanagida K. Yazawa H. Katayose H. Kimura Y. Hayashi S. Sato A. Oocyte activation induced by spermatids and the spermatozoa.Int J Androl. 2000; 23: 63-65Crossref PubMed Scopus (17) Google Scholar). Extraction and delivery of this factor or treatment with a calcium ionophore are two potential strategies for inducing oocyte activation, which is required for fertilization.Embryonic developmentIn primate species, the embryonic centrosome normally derives from spindle elements contributed by spermatozoa. Because round spermatids have not yet developed mature centriolar complexes with spindles, their injection into oocytes has the theoretical potential for unexpected and possibly adverse effects on subsequent embryonic development.Genetic abnormalitiesOur current understanding of the molecular control mechanisms that regulate spermatogenesis, sperm transport, fertilization, and early embryonic development is severely limited. Consequently, nearly one quarter of all male infertility is categorized as idiopathic. Among men with nonobstructive azoospermia who are candidates for TESE, only approximately 17% have identifiable genetic abnormalities. Most affected men have chromosomal anomalies, predominantly Klinefelter syndrome (46,XXY); 7% to 10% harbor known Y chromosome microdeletions (16Rucker G.B. Mielnik A. King P. Goldstein M. Schlegel P.N. Preoperative screening for genetic abnormalities in men with nonobstructive azoospermia before testicular sperm extraction.J Urol. 1998; 160: 2068-2071Abstract Full Text Full Text PDF PubMed Google Scholar).Among remaining azoospermic men, some are likely to have as yet unidentified autosomal genetic defects (17Lamb D.J. Debate is ICSI a genetic time bomb? Yes.J Androl. 1999; 20: 23-33PubMed Google Scholar). Certainly, any genetic abnormality sufficiently severe to result in meiotic arrest (the only cause of infertility that could possibly benefit from application of ROSNI) might also have adverse effects on other normal cellular processes or other systemic manifestations. Offspring conceived via IVF with ROSNI may thus be at risk for infertility or even more severe genetic defects.The health consequences for offspring conceived through ROSNI are largely unknown. An early report suggested that IVF with ICSI might be associated with an increased risk for sex chromosome abnormalities (18Bonduelle M. Aytoz A. Van Assche E. Devroey P. Liebaers I. Van Steirteghem A. Incidence of chromosomal aberrations in children born after assisted reproduction through intracytoplasmic sperm injection.Hum Reprod. 1998; 13: 781-782Crossref PubMed Scopus (184) Google Scholar). Subsequent studies have suggested that ICSI has no intrinsic genetic risks (19Bonduelle M. Camus M. De Vos A. Staessen C. Tournaye H. Van Assche E. et al.Seven years of intracytoplasmic sperm injection and follow-up of 1987 subsequent children.Hum Reprod. 1999; 14: 243-264Crossref PubMed Scopus (216) Google Scholar, 20Bonduelle M. Wilikens A. Buysse A. et al.A follow-up study of children born after intracytoplasmic sperm injection (ICSI) with epididymal and testicular spermatozoa and after replacement of cryopreserved embryos obtained after ICSI.Hum Reprod. 1998; 13: 196-207Crossref PubMed Scopus (91) Google Scholar, 21Tarlatzis B.C. Report on the activities of the ESHRE Task Force on intracytoplasmic sperm injection. European Society of Human Reproduction and Embryology.Hum Reprod. 1996; 11: 160-185Crossref PubMed Scopus (29) Google Scholar) or that risk of birth defects is increased with IVF, but to no greater extent when ICSI is also performed using ejaculated sperm (22Hansen M. Kurinczuk J.J. Bower C. Webb S. The risk of major birth defects after intracytoplasmic sperm injection and in vitro fertilization.N Engl J Med. 2002; 346: 725-730Crossref PubMed Scopus (917) Google Scholar). However, data regarding the safety of ICSI using testicular sperm or spermatids obtained from men with nonobstructive azoospermia are far more limited.Genomic imprinting (modification of gene expression by cytosine methylation) normally occurs during gametogenesis and may not be complete early in the round spermatid stage of development. Naturally occurring errors in imprinting may result in illness, such as type II Angelman syndrome (a genetic disorder characterized by mental retardation, lack of speech, and movement and behavior disorders). In mice, genomic imprinting is completed in the round spermatid (23Shamanski F.L. Kimura Y. Lavoir M.C. Pedersen R.A. Yanagimachi R. Status of genomic imprinting in mouse spermatids.Hum Reprod. 1999; 14: 1050-1056Crossref PubMed Scopus (78) Google Scholar), but the exact stage of genomic imprinting in human spermatogenesis is unknown and must be defined before clinical application of ROSNI in human IVF.Limited success ratesTo date, the application of ROSNI in clinical IVF has had disappointing results. Although several reports have suggested the feasibility of ROSNI in human IVF (8Gianaroli L. Selman H.A. Magli M.C. Colpi G. Fortini D. Ferraretti A.P. Birth of a healthy infant after conception with round spermatids isolated from cryopreserved testicular tissue.Fertil Steril. 1999; 72: 539-541Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 9Tesarik J. Cruz-Navarro N. Moreno E. Canete M.T. Mendoza C. Birth of healthy twins after fertilization with in vitro cultured spermatids from a patient with massive in vivo apoptosis of postmeiotic germ cells.Fertil Steril. 2000; 74: 1044-1046Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 10Schoysman R. Vanderzwalmen P. Bertin G. Nijs M. Van Damme B. Oocyte insemination with spermatozoa precursors.Curr Opin Urol. 1999; 9: 541-545Crossref PubMed Scopus (12) Google Scholar, 24Al-Hasani S. Ludwig M. Palermo I. et al.Intracytoplasmic injection of round and elongated spermatids from azoospermic patients results and review.Hum Reprod. 1999; 14: 97-107Crossref PubMed Scopus (35) Google Scholar, 25Choavaratana R. Suppinyopong S. Chaimahaphruksa P. ROSI from TESE the first case in Thailand a case report.J Med Assoc Thai. 1999; 82: 938-941PubMed Google Scholar), overall fertilization rates achieved with ROSNI are lower (45% to 50%) than with ICSI using mature sperm or elongating spermatids (69% to 74%) (10Schoysman R. Vanderzwalmen P. Bertin G. Nijs M. Van Damme B. Oocyte insemination with spermatozoa precursors.Curr Opin Urol. 1999; 9: 541-545Crossref PubMed Scopus (12) Google Scholar, 26Vicdan K. Isik A.Z. Delilbasi L. Development of blastocyst-stage embryos after round spermatid injection in patients with complete spermiogenesis failure.J Assist Reprod Genet. 2001; 18: 78-86Crossref PubMed Scopus (29) Google Scholar, 27Levran D. Nahum H. Farhi J. Weissman A. Poor outcome with round spermatid injection in azoospermic patients with maturation arrest.Fertil Steril. 2000; 74: 443-449Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 28Balaban B. Urman B. Isiklar A. Alatas C. Aksoy S. Mercan R. et al.Progression to the blastocyst stage of embryos derived from testicular round spermatids.Hum Reprod. 2000; 15: 1377-1382Crossref PubMed Scopus (44) Google Scholar). One report on the occurrence of significant congenital anomalies in ROSNI-conceived pregnancies raises further concerns (29Zech H. Vanderzwalmen P. Prapas Y. Lejeune B. Duba E. Schoysman R. Congenital malformations after intracytoplasmic injection of spermatids.Hum Reprod. 2000; 15: 969-971Crossref PubMed Scopus (58) Google Scholar). Although causality was not established, the report indicates that caution is warranted. Round spermatid identificationROSNI has been used to achieve fertilization in human IVF, but with limited success (8Gianaroli L. Selman H.A. Magli M.C. Colpi G. Fortini D. Ferraretti A.P. Birth of a healthy infant after conception with round spermatids isolated from cryopreserved testicular tissue.Fertil Steril. 1999; 72: 539-541Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 9Tesarik J. Cruz-Navarro N. Moreno E. Canete M.T. Mendoza C. Birth of healthy twins after fertilization with in vitro cultured spermatids from a patient with massive in vivo apoptosis of postmeiotic germ cells.Fertil Steril. 2000; 74: 1044-1046Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 10Schoysman R. Vanderzwalmen P. Bertin G. Nijs M. Van Damme B. Oocyte insemination with spermatozoa precursors.Curr Opin Urol. 1999; 9: 541-545Crossref PubMed Scopus (12) Google Scholar). Accurate identification of round spermatid cells is one technical challenge of ROSNI. Using the standard optics present in most clinical IVF laboratories, it can be difficult to distinguish haploid round spermatid cells from diploid spermatogenic precursors and somatic cells. Phase contrast microscopy may simplify the task (11Verheyen G. Crabbe E. Joris H. Van Steirteghem A. Simple and reliable identification of the human round spermatid by inverted phase-contrast microscopy.Hum Reprod. 1998; 13: 1570-1577Crossref PubMed Scopus (34) Google Scholar). Another technique recently described uses a fluorescent mitochondrial probe to specifically identify round spermatids in dispersed testicular cells (12Sutovsky P. Ramalho-Santos J. Moreno R.D. Oko R. Hewitson L. Schatten G. On-stage selection of single round spermatids using a vital, mitochondrion-specific fluorescent probe MitoTracker(TM) and high resolution differential interference contrast microscopy.Hum Reprod. 1999; 14: 2301-2312Crossref PubMed Scopus (43) Google Scholar). Originally developed for use in nonhuman primate species and cattle, the technique will require extensive testing to establish its safety before it can be applied in humans. ROSNI has been used to achieve fertilization in human IVF, but with limited success (8Gianaroli L. Selman H.A. Magli M.C. Colpi G. Fortini D. Ferraretti A.P. Birth of a healthy infant after conception with round spermatids isolated from cryopreserved testicular tissue.Fertil Steril. 1999; 72: 539-541Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 9Tesarik J. Cruz-Navarro N. Moreno E. Canete M.T. Mendoza C. Birth of healthy twins after fertilization with in vitro cultured spermatids from a patient with massive in vivo apoptosis of postmeiotic germ cells.Fertil Steril. 2000; 74: 1044-1046Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 10Schoysman R. Vanderzwalmen P. Bertin G. Nijs M. Van Damme B. Oocyte insemination with spermatozoa precursors.Curr Opin Urol. 1999; 9: 541-545Crossref PubMed Scopus (12) Google Scholar). Accurate identification of round spermatid cells is one technical challenge of ROSNI. Using the standard optics present in most clinical IVF laboratories, it can be difficult to distinguish haploid round spermatid cells from diploid spermatogenic precursors and somatic cells. Phase contrast microscopy may simplify the task (11Verheyen G. Crabbe E. Joris H. Van Steirteghem A. Simple and reliable identification of the human round spermatid by inverted phase-contrast microscopy.Hum Reprod. 1998; 13: 1570-1577Crossref PubMed Scopus (34) Google Scholar). Another technique recently described uses a fluorescent mitochondrial probe to specifically identify round spermatids in dispersed testicular cells (12Sutovsky P. Ramalho-Santos J. Moreno R.D. Oko R. Hewitson L. Schatten G. On-stage selection of single round spermatids using a vital, mitochondrion-specific fluorescent probe MitoTracker(TM) and high resolution differential interference contrast microscopy.Hum Reprod. 1999; 14: 2301-2312Crossref PubMed Scopus (43) Google Scholar). Originally developed for use in nonhuman primate species and cattle, the technique will require extensive testing to establish its safety before it can be applied in humans. Oocyte activationActivation of the oocyte rarely occurs after spermatid injection alone. This observation suggests that another factor present in mature spermatozoa may serve to induce a calcium flux in the oocyte, resulting in its activation (13Tesarik J. Mendoza C. Greco E. The activity (calcium oscillator?) responsible for human oocyte activation after injection with round spermatids is associated with spermatid nuclei.Fertil Steril. 2000; 74: 1245-1247Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 14Yazawa H. Yanagida K. Katayose H. Hayashi S. Sato A. Comparison of oocyte activation and Ca2+ oscillation-inducing abilities of round/elongated spermatids of mouse, hamster, rat, rabbit and human assessed by mouse oocyte activation assay.Hum Reprod. 2000; 15: 2582-2590Crossref PubMed Scopus (51) Google Scholar, 15Yanagida K. Yazawa H. Katayose H. Kimura Y. Hayashi S. Sato A. Oocyte activation induced by spermatids and the spermatozoa.Int J Androl. 2000; 23: 63-65Crossref PubMed Scopus (17) Google Scholar). Extraction and delivery of this factor or treatment with a calcium ionophore are two potential strategies for inducing oocyte activation, which is required for fertilization. Activation of the oocyte rarely occurs after spermatid injection alone. This observation suggests that another factor present in mature spermatozoa may serve to induce a calcium flux in the oocyte, resulting in its activation (13Tesarik J. Mendoza C. Greco E. The activity (calcium oscillator?) responsible for human oocyte activation after injection with round spermatids is associated with spermatid nuclei.Fertil Steril. 2000; 74: 1245-1247Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 14Yazawa H. Yanagida K. Katayose H. Hayashi S. Sato A. Comparison of oocyte activation and Ca2+ oscillation-inducing abilities of round/elongated spermatids of mouse, hamster, rat, rabbit and human assessed by mouse oocyte activation assay.Hum Reprod. 2000; 15: 2582-2590Crossref PubMed Scopus (51) Google Scholar, 15Yanagida K. Yazawa H. Katayose H. Kimura Y. Hayashi S. Sato A. Oocyte activation induced by spermatids and the spermatozoa.Int J Androl. 2000; 23: 63-65Crossref PubMed Scopus (17) Google Scholar). Extraction and delivery of this factor or treatment with a calcium ionophore are two potential strategies for inducing oocyte activation, which is required for fertilization. Embryonic developmentIn primate species, the embryonic centrosome normally derives from spindle elements contributed by spermatozoa. Because round spermatids have not yet developed mature centriolar complexes with spindles, their injection into oocytes has the theoretical potential for unexpected and possibly adverse effects on subsequent embryonic development. In primate species, the embryonic centrosome normally derives from spindle elements contributed by spermatozoa. Because round spermatids have not yet developed mature centriolar complexes with spindles, their injection into oocytes has the theoretical potential for unexpected and possibly adverse effects on subsequent embryonic development. Genetic abnormalitiesOur current understanding of the molecular control mechanisms that regulate spermatogenesis, sperm transport, fertilization, and early embryonic development is severely limited. Consequently, nearly one quarter of all male infertility is categorized as idiopathic. Among men with nonobstructive azoospermia who are candidates for TESE, only approximately 17% have identifiable genetic abnormalities. Most affected men have chromosomal anomalies, predominantly Klinefelter syndrome (46,XXY); 7% to 10% harbor known Y chromosome microdeletions (16Rucker G.B. Mielnik A. King P. Goldstein M. Schlegel P.N. Preoperative screening for genetic abnormalities in men with nonobstructive azoospermia before testicular sperm extraction.J Urol. 1998; 160: 2068-2071Abstract Full Text Full Text PDF PubMed Google Scholar).Among remaining azoospermic men, some are likely to have as yet unidentified autosomal genetic defects (17Lamb D.J. Debate is ICSI a genetic time bomb? Yes.J Androl. 1999; 20: 23-33PubMed Google Scholar). Certainly, any genetic abnormality sufficiently severe to result in meiotic arrest (the only cause of infertility that could possibly benefit from application of ROSNI) might also have adverse effects on other normal cellular processes or other systemic manifestations. Offspring conceived via IVF with ROSNI may thus be at risk for infertility or even more severe genetic defects.The health consequences for offspring conceived through ROSNI are largely unknown. An early report suggested that IVF with ICSI might be associated with an increased risk for sex chromosome abnormalities (18Bonduelle M. Aytoz A. Van Assche E. Devroey P. Liebaers I. Van Steirteghem A. Incidence of chromosomal aberrations in children born after assisted reproduction through intracytoplasmic sperm injection.Hum Reprod. 1998; 13: 781-782Crossref PubMed Scopus (184) Google Scholar). Subsequent studies have suggested that ICSI has no intrinsic genetic risks (19Bonduelle M. Camus M. De Vos A. Staessen C. Tournaye H. Van Assche E. et al.Seven years of intracytoplasmic sperm injection and follow-up of 1987 subsequent children.Hum Reprod. 1999; 14: 243-264Crossref PubMed Scopus (216) Google Scholar, 20Bonduelle M. Wilikens A. Buysse A. et al.A follow-up study of children born after intracytoplasmic sperm injection (ICSI) with epididymal and testicular spermatozoa and after replacement of cryopreserved embryos obtained after ICSI.Hum Reprod. 1998; 13: 196-207Crossref PubMed Scopus (91) Google Scholar, 21Tarlatzis B.C. Report on the activities of the ESHRE Task Force on intracytoplasmic sperm injection. European Society of Human Reproduction and Embryology.Hum Reprod. 1996; 11: 160-185Crossref PubMed Scopus (29) Google Scholar) or that risk of birth defects is increased with IVF, but to no greater extent when ICSI is also performed using ejaculated sperm (22Hansen M. Kurinczuk J.J. Bower C. Webb S. The risk of major birth defects after intracytoplasmic sperm injection and in vitro fertilization.N Engl J Med. 2002; 346: 725-730Crossref PubMed Scopus (917) Google Scholar). However, data regarding the safety of ICSI using testicular sperm or spermatids obtained from men with nonobstructive azoospermia are far more limited.Genomic imprinting (modification of gene expression by cytosine methylation) normally occurs during gametogenesis and may not be complete early in the round spermatid stage of development. Naturally occurring errors in imprinting may result in illness, such as type II Angelman syndrome (a genetic disorder characterized by mental retardation, lack of speech, and movement and behavior disorders). In mice, genomic imprinting is completed in the round spermatid (23Shamanski F.L. Kimura Y. Lavoir M.C. Pedersen R.A. Yanagimachi R. Status of genomic imprinting in mouse spermatids.Hum Reprod. 1999; 14: 1050-1056Crossref PubMed Scopus (78) Google Scholar), but the exact stage of genomic imprinting in human spermatogenesis is unknown and must be defined before clinical application of ROSNI in human IVF. Our current understanding of the molecular control mechanisms that regulate spermatogenesis, sperm transport, fertilization, and early embryonic development is severely limited. Consequently, nearly one quarter of all male infertility is categorized as idiopathic. Among men with nonobstructive azoospermia who are candidates for TESE, only approximately 17% have identifiable genetic abnormalities. Most affected men have chromosomal anomalies, predominantly Klinefelter syndrome (46,XXY); 7% to 10% harbor known Y chromosome microdeletions (16Rucker G.B. Mielnik A. King P. Goldstein M. Schlegel P.N. Preoperative screening for genetic abnormalities in men with nonobstructive azoospermia before testicular sperm extraction.J Urol. 1998; 160: 2068-2071Abstract Full Text Full Text PDF PubMed Google Scholar). Among remaining azoospermic men, some are likely to have as yet unidentified autosomal genetic defects (17Lamb D.J. Debate is ICSI a genetic time bomb? Yes.J Androl. 1999; 20: 23-33PubMed Google Scholar). Certainly, any genetic abnormality sufficiently severe to result in meiotic arrest (the only cause of infertility that could possibly benefit from application of ROSNI) might also have adverse effects on other normal cellular processes or other systemic manifestations. Offspring conceived via IVF with ROSNI may thus be at risk for infertility or even more severe genetic defects. The health consequences for offspring conceived through ROSNI are largely unknown. An early report suggested that IVF with ICSI might be associated with an increased risk for sex chromosome abnormalities (18Bonduelle M. Aytoz A. Van Assche E. Devroey P. Liebaers I. Van Steirteghem A. Incidence of chromosomal aberrations in children born after assisted reproduction through intracytoplasmic sperm injection.Hum Reprod. 1998; 13: 781-782Crossref PubMed Scopus (184) Google Scholar). Subsequent studies have suggested that ICSI has no intrinsic genetic risks (19Bonduelle M. Camus M. De Vos A. Staessen C. Tournaye H. Van Assche E. et al.Seven years of intracytoplasmic sperm injection and follow-up of 1987 subsequent children.Hum Reprod. 1999; 14: 243-264Crossref PubMed Scopus (216) Google Scholar, 20Bonduelle M. Wilikens A. Buysse A. et al.A follow-up study of children born after intracytoplasmic sperm injection (ICSI) with epididymal and testicular spermatozoa and after replacement of cryopreserved embryos obtained after ICSI.Hum Reprod. 1998; 13: 196-207Crossref PubMed Scopus (91) Google Scholar, 21Tarlatzis B.C. Report on the activities of the ESHRE Task Force on intracytoplasmic sperm injection. European Society of Human Reproduction and Embryology.Hum Reprod. 1996; 11: 160-185Crossref PubMed Scopus (29) Google Scholar) or that risk of birth defects is increased with IVF, but to no greater extent when ICSI is also performed using ejaculated sperm (22Hansen M. Kurinczuk J.J. Bower C. Webb S. The risk of major birth defects after intracytoplasmic sperm injection and in vitro fertilization.N Engl J Med. 2002; 346: 725-730Crossref PubMed Scopus (917) Google Scholar). However, data regarding the safety of ICSI using testicular sperm or spermatids obtained from men with nonobstructive azoospermia are far more limited. Genomic imprinting (modification of gene expression by cytosine methylation) normally occurs during gametogenesis and may not be complete early in the round spermatid stage of development. Naturally occurring errors in imprinting may result in illness, such as type II Angelman syndrome (a genetic disorder characterized by mental retardation, lack of speech, and movement and behavior disorders). In mice, genomic imprinting is completed in the round spermatid (23Shamanski F.L. Kimura Y. Lavoir M.C. Pedersen R.A. Yanagimachi R. Status of genomic imprinting in mouse spermatids.Hum Reprod. 1999; 14: 1050-1056Crossref PubMed Scopus (78) Google Scholar), but the exact stage of genomic imprinting in human spermatogenesis is unknown and must be defined before clinical application of ROSNI in human IVF. Limited success ratesTo date, the application of ROSNI in clinical IVF has had disappointing results. Although several reports have suggested the feasibility of ROSNI in human IVF (8Gianaroli L. Selman H.A. Magli M.C. Colpi G. Fortini D. Ferraretti A.P. Birth of a healthy infant after conception with round spermatids isolated from cryopreserved testicular tissue.Fertil Steril. 1999; 72: 539-541Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 9Tesarik J. Cruz-Navarro N. Moreno E. Canete M.T. Mendoza C. Birth of healthy twins after fertilization with in vitro cultured spermatids from a patient with massive in vivo apoptosis of postmeiotic germ cells.Fertil Steril. 2000; 74: 1044-1046Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 10Schoysman R. Vanderzwalmen P. Bertin G. Nijs M. Van Damme B. Oocyte insemination with spermatozoa precursors.Curr Opin Urol. 1999; 9: 541-545Crossref PubMed Scopus (12) Google Scholar, 24Al-Hasani S. Ludwig M. Palermo I. et al.Intracytoplasmic injection of round and elongated spermatids from azoospermic patients results and review.Hum Reprod. 1999; 14: 97-107Crossref PubMed Scopus (35) Google Scholar, 25Choavaratana R. Suppinyopong S. Chaimahaphruksa P. ROSI from TESE the first case in Thailand a case report.J Med Assoc Thai. 1999; 82: 938-941PubMed Google Scholar), overall fertilization rates achieved with ROSNI are lower (45% to 50%) than with ICSI using mature sperm or elongating spermatids (69% to 74%) (10Schoysman R. Vanderzwalmen P. Bertin G. Nijs M. Van Damme B. Oocyte insemination with spermatozoa precursors.Curr Opin Urol. 1999; 9: 541-545Crossref PubMed Scopus (12) Google Scholar, 26Vicdan K. Isik A.Z. Delilbasi L. Development of blastocyst-stage embryos after round spermatid injection in patients with complete spermiogenesis failure.J Assist Reprod Genet. 2001; 18: 78-86Crossref PubMed Scopus (29) Google Scholar, 27Levran D. Nahum H. Farhi J. Weissman A. Poor outcome with round spermatid injection in azoospermic patients with maturation arrest.Fertil Steril. 2000; 74: 443-449Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 28Balaban B. Urman B. Isiklar A. Alatas C. Aksoy S. Mercan R. et al.Progression to the blastocyst stage of embryos derived from testicular round spermatids.Hum Reprod. 2000; 15: 1377-1382Crossref PubMed Scopus (44) Google Scholar). One report on the occurrence of significant congenital anomalies in ROSNI-conceived pregnancies raises further concerns (29Zech H. Vanderzwalmen P. Prapas Y. Lejeune B. Duba E. Schoysman R. Congenital malformations after intracytoplasmic injection of spermatids.Hum Reprod. 2000; 15: 969-971Crossref PubMed Scopus (58) Google Scholar). Although causality was not established, the report indicates that caution is warranted. To date, the application of ROSNI in clinical IVF has had disappointing results. Although several reports have suggested the feasibility of ROSNI in human IVF (8Gianaroli L. Selman H.A. Magli M.C. Colpi G. Fortini D. Ferraretti A.P. Birth of a healthy infant after conception with round spermatids isolated from cryopreserved testicular tissue.Fertil Steril. 1999; 72: 539-541Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 9Tesarik J. Cruz-Navarro N. Moreno E. Canete M.T. Mendoza C. Birth of healthy twins after fertilization with in vitro cultured spermatids from a patient with massive in vivo apoptosis of postmeiotic germ cells.Fertil Steril. 2000; 74: 1044-1046Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 10Schoysman R. Vanderzwalmen P. Bertin G. Nijs M. Van Damme B. Oocyte insemination with spermatozoa precursors.Curr Opin Urol. 1999; 9: 541-545Crossref PubMed Scopus (12) Google Scholar, 24Al-Hasani S. Ludwig M. Palermo I. et al.Intracytoplasmic injection of round and elongated spermatids from azoospermic patients results and review.Hum Reprod. 1999; 14: 97-107Crossref PubMed Scopus (35) Google Scholar, 25Choavaratana R. Suppinyopong S. Chaimahaphruksa P. ROSI from TESE the first case in Thailand a case report.J Med Assoc Thai. 1999; 82: 938-941PubMed Google Scholar), overall fertilization rates achieved with ROSNI are lower (45% to 50%) than with ICSI using mature sperm or elongating spermatids (69% to 74%) (10Schoysman R. Vanderzwalmen P. Bertin G. Nijs M. Van Damme B. Oocyte insemination with spermatozoa precursors.Curr Opin Urol. 1999; 9: 541-545Crossref PubMed Scopus (12) Google Scholar, 26Vicdan K. Isik A.Z. Delilbasi L. Development of blastocyst-stage embryos after round spermatid injection in patients with complete spermiogenesis failure.J Assist Reprod Genet. 2001; 18: 78-86Crossref PubMed Scopus (29) Google Scholar, 27Levran D. Nahum H. Farhi J. Weissman A. Poor outcome with round spermatid injection in azoospermic patients with maturation arrest.Fertil Steril. 2000; 74: 443-449Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 28Balaban B. Urman B. Isiklar A. Alatas C. Aksoy S. Mercan R. et al.Progression to the blastocyst stage of embryos derived from testicular round spermatids.Hum Reprod. 2000; 15: 1377-1382Crossref PubMed Scopus (44) Google Scholar). One report on the occurrence of significant congenital anomalies in ROSNI-conceived pregnancies raises further concerns (29Zech H. Vanderzwalmen P. Prapas Y. Lejeune B. Duba E. Schoysman R. Congenital malformations after intracytoplasmic injection of spermatids.Hum Reprod. 2000; 15: 969-971Crossref PubMed Scopus (58) Google Scholar). Although causality was not established, the report indicates that caution is warranted. Summary and recommendations·ROSNI is a method of assisted fertilization in which precursors of mature spermatozoa are injected into oocytes.·Accurate identification of round spermatids remains a technical challenge.·Other important unresolved issues include impaired oocyte activation, potential adverse effects on the embryonic centrosome, and risk of infertility or more severe genetic defects in offspring.·The health consequences of ROSNI for offspring are uncertain.·Further research is required to better define the genetic causes of male infertility and to establish the safety of ROSNI and other novel assisted reproductive technologies.·ROSNI should not be performed when more mature sperm forms (elongating spermatids or spermatozoa) can be identified and used for ICSI.·Patients who may be candidates for ROSNI should receive careful and thorough pretreatment counseling to ensure they are clearly informed of the limitations and potential risks of the procedure.·Application of ROSNI in clinical human IVF should be considered experimental and therefore requires approval and oversight by an appropriately constituted Institutional Review Board. ·ROSNI is a method of assisted fertilization in which precursors of mature spermatozoa are injected into oocytes.·Accurate identification of round spermatids remains a technical challenge.·Other important unresolved issues include impaired oocyte activation, potential adverse effects on the embryonic centrosome, and risk of infertility or more severe genetic defects in offspring.·The health consequences of ROSNI for offspring are uncertain.·Further research is required to better define the genetic causes of male infertility and to establish the safety of ROSNI and other novel assisted reproductive technologies.·ROSNI should not be performed when more mature sperm forms (elongating spermatids or spermatozoa) can be identified and used for ICSI.·Patients who may be candidates for ROSNI should receive careful and thorough pretreatment counseling to ensure they are clearly informed of the limitations and potential risks of the procedure.·Application of ROSNI in clinical human IVF should be considered experimental and therefore requires approval and oversight by an appropriately constituted Institutional Review Board.